CN104487587A

CN104487587A - Cd3 binding polypeptides

Info

Publication number: CN104487587A
Application number: CN201380027109.8A
Authority: CN
Inventors: P·H·坦; S·K·纳塔拉詹; C·J·麦克马汉
Original assignee: Emergent Product Development Seattle LLC
Current assignee: Aptevo Research and Development LLC
Priority date: 2012-04-20
Filing date: 2013-04-18
Publication date: 2015-04-01
Also published as: US20150232557A1; HK1208707A1; RU2014142166A; EP2839019A4; BR112014025830A2; MX2014012578A; IN2014MN02293A; WO2013158856A2; AU2017228643B2; CA2870545A1; MX366813B; EP2839019A2; US10202452B2; HK1207398A1; KR20150004856A; RU2673153C2; SG10201608307WA; JP6509724B2; JP2015521032A; IL235093A0

Abstract

The present invention relates to mono-specific and multi-specific protein therapeutics that bind or interact with CD3. This includes antibodies, fragments thereof, scFvs, Fabs, di-scFvs single domain antibodies, diabodies, dual variable domain binding proteins and polypeptides containing an antibody or antibody fragments. In one embodiment, multispecific polypeptides that bind to T-cells receptor complex on T-cells and tumor cells to induce target-dependent T-cell cytotoxicity, activation and proliferation,.

Description

CD3 Binding peptide

Related application

The present invention relates to the PCT application PCT/US12/034575 that the title submitted on April 20th, 2012 is " Prostate-SpecificMembrane Antigen Binding Proteins And Related Compositions AndMethods ", described PCT application requires the U.S. Provisional Patent Application 61/478 that on April 22nd, 2011 submits to, the right of priority of 449, and also relate to U. S. application number 61/636,557, the mode that these three documents are all quoted in full is incorporated to.

Invention field

The present invention relates in conjunction with CD3 or monospecific interactional with it and the agent of polyspecific protein for treatment.This polypeptide comprising antibody, its fragment, scFv, Fab, two scFv single domain antibody and contain antibody or antibody fragment.

To enclose sequence table

Text (title: " 2479_113PC03_SequenceListing_ascii.txt ", size: 459,722 bytes Electronically submitted to together with herein; Date created: on April 16th, 2013) the mode quoted in full of content be incorporated herein.

Background

With the TCR mixture in CD 3-resisting monoclonal antibody targeted human T cell for or propose be used for the treatment of autoimmune disorders and associated conditions, be such as used for the treatment of organ allograft repel.To the specific mouse monoclonal antibody of people CD3, such as OKT3 (Kung etc. (1979) Science 206:347-9) is the first-generation of described treatment.Although OKT3 has strong immunosuppression usefulness, the serious side effects relevant with mitogenesis potentiality to its immunogenicity hampers its Clinical practice (Chatenoud (2003) Nature Reviews 3:123-132).It induces antiglobulin reaction, thus promotes himself to remove fast and neutralization (Chatenoud etc. (1982) Eur.J.Immunol.137:830-8).In addition, OKT3 in vitro inducing T cell proliferation and cytokine produces, and causes the extensive release (Hirsch etc. (1989) J.Immunol 142:737-43,1989) of cytokine in vivo.Release of cytokines (cause again having a fever also referred to as " cytokine storm ", shiver with cold, " influenza sample " syndrome (Chatenoud, 2003) that headache, nausea,vomiting,diarrhea, respiratory distress, aseptic meningitis and hypopiesia are feature.Described serious side effects limits OKT3 broadly for expanding to other clinical field, such as autoimmune (the same) in transplanting and by its purposes.

In order to reduce the side effect of first-generation CD 3-resisting monoclonal antibody, complementarity-determining region (CDR) is transplanted in human IgG sequence not by means of only muroid CD 3-resisting monoclonal antibody, and have developed the s-generation through genetic engineering modified CD 3-resisting monoclonal antibody (Cole etc. (1999) Transplantation 68:563 by being combined to suddenly change to be incorporated in Fc by non-FcR to reduce the generation of cytokine storm; Cole etc. (1997) J.Immunol.159:3613).Also see PCT publication No. WO2010/042904, the mode that the document is quoted in full is incorporated herein.

Except the monospecific therapeutical agent of target CD3, the polyspecific polypeptide optionally in conjunction with T cell and tumour cell also can provide redirected T cell cytotoxicity with the mechanism for tumour cell and Therapeutic cancer.But the problem that design dual specific or polyspecific T cell raise antibody is maintain specificity and cross T cell activation rule by multiple adjustment path simultaneously.

Still AntiCD3 McAb monospecific and the multispecific molecule of improvement is needed.Although previously carried out improveing with the possibility reducing cytokine storm to the Fc part of CD3 targeted molecular, but still the AntiCD3 McAb therapeutical agent of redirected T cell cytotoxicity (when being designed for the multispecific molecule of Therapeutic cancer) that transformation period, the T cell that effectively can manufacture, represent improvement that needs have increase compared with prior art molecule combine and/or improve.

Summary of the invention

In one aspect, the present invention includes a kind of CD3 Binding peptide, it comprises the CD3 binding domain compared with having the binding domain of aminoacid sequence SEQ ID NO:41 with the iso-electric point of reduction.In certain embodiments, described CD3 binding domain comprises VH and VL district, VH and VL district is separately containing framework region.In order to reduce the iso-electric point (pI) of described binding domain and/or polypeptide, can by replacing the amino acid of positively charged and/or two or more amino acid of modifying with electronegative aminoacid replacement neutral amino acids in framework region with neutral amino acids.For example, R and T can be replaced with S, Q can be replaced with E, Y can be replaced with F, and T can be replaced with V.In one embodiment, such as, in order to reduce immunogenic risk, the amino acid substituted onto in sequence is ubiquitous in human germ-line system IgG sequence or is included in the identical or contiguous position in human germ-line system sequence, such as, in the human germ-line system IgG sequence of correspondence.In some embodiments, described human germ-line system IgG sequence comprises SEQ ID NO:43 or 44.

In another embodiment of the invention, described CD3 Binding peptide comprises the modification of the pI that can reduce described front hinge area or whole polypeptide in front hinge area (prehinge region).Described front hinge area is in the joint between described binding domain and hinge area.For example, the front hinge area of triamino acid of sequence RRT can be had to reduce iso-electric point with sequence SSS displacement.In some embodiments, front hinge area has the iso-electric point of reduction compared with having the CD3 Binding peptide of the front hinge area of RRT.

CD3 Binding peptide can be designed such as to reduce iso-electric point by making the framework region of VH and/or VL district and/or front hinge area suddenly change.Iso-electric point can reduce by 0.5 to 2.5 or more units.

In one embodiment of the invention, described CD3 Binding peptide is antibody, such as humanized antibody.In another embodiment, it is a kind of little module formula immune drug albumen (SMIP).In some embodiments, CD3 Binding peptide comprises single chain variable fragment (scFv).

In another embodiment, it is polyspecific or dual specific polypeptide.For example, described CD3 Binding peptide can comprise CD3 binding domain and tumour antigen binding domain.In one embodiment, described dual specific or multispecific molecule are redirected T cell cytotoxicity with for tumour cell.

In another embodiment of the invention, described CD3 polypeptide exists as homodimer or heterodimer.

Accompanying drawing is sketched

Fig. 1 is the figure of theoretical pI compared with the theoretical pI of about 200 kinds of other SMIP polypeptide describing the humanization Cris-7SMIP polypeptide previously produced.

Fig. 2 A and 2B describe comprise the pI higher than CD3 polypeptide of the present invention CD3 in conjunction with the montage of SMIP polypeptide.Fig. 2 A is the SDS capillary electrophoresis of the lower molecular weight montage section content of the quantitative purified protein of energy.Fig. 2 B contains the explanation of montage type CD3SMIP molecule.Sequence shown in Fig. 2 B corresponds to the 251-260 of 243-252 and the SEQ ID NO:240 of amino acid 252-260, SEQ ID NO:4 of SEQ ID NO:4.

Fig. 3 is the amino acid alignment of Cris-7 mouse VH and derivative humanized VH sequence.Cris7VH mouse, H1, H2, H3, H4, H5, H6 and consensus sequence correspond respectively to SEQ ID NO:45,22,24,26,28,30,32 and 229.

Fig. 4 is the amino acid alignment of Cris-7 mouse VL and derivative humanization VL sequence.Cris7VL mouse, L1, L2, L3, L4 and consensus sequence correspond respectively to SEQ IDNO:46,34,36,38,40 and 230.

Fig. 5 is the amino acid alignment of H3 and germline sequence IGHV1-69*01 and IGHV3-30*01.H3, IGHV1-69*01, IGHV3-30*01 and consensus sequence correspond respectively to SEQ ID NO:26,43,44 and 331.

Fig. 6 is the amino acid alignment of J κ district IGKV1-5*01 and the L1 in the VL district of germline sequence.The J κ district (IGKVI-5*01) in described VL district, L1 chain and consensus sequence correspond respectively to SEQ ID NO:232,34 and 233.Listed IGKJ1*01, IGKJ2*01, IGKJ3*01, IGKJ4*01 and IGKJ5*01 sequence corresponds respectively to SEQ IDNO:234-238.

Fig. 7 A with 7B depicts the amino acid alignment of the various CD3 Binding peptide SMIP molecules of the theoretical pI compared with DRA209 with reduction.Amino acid/11-423, the amino acid/11-423 of SEQ ID NO:6, the amino acid/11-423 of SEQ ID NO:8, the amino acid/11-423 of SEQ ID NO:10, the amino acid/11-423 of SEQ ID NO:12, the amino acid/11-426 of SEQ ID NO:14, the amino acid/11-426 of SEQ ID NO:16 and SEQ ID NO:239 of SEQ ID NO:4 is corresponded respectively to for the sequence listed by DRA209, DRA219, DRA222, DRA221, DRA223, DRA224, DRA225 and consensus sequence.

Fig. 8 is the amino acid alignment that pI modification D RA233 compares with DRA234 and DRA161.SEQ ID NO:240,18,20 and 241 is corresponded respectively to for the sequence listed by DRA161, DRA233, DRA234 and consensus sequence.

Fig. 9 is the explanation how determining experience pI value.

Figure 10 is the figure of the experience pI of display DRA209SMIP and scFv.

Figure 11 is the figure of the experience pI that display DRA161SMIP and pI modification D RA233 with DRA234 compares.

Figure 12 is the figure of the display mode of identical with DRA209 with DRA161 (except DRA227) in conjunction with the SMIP pI variant of T cell.DRA227SMIP is with to compare and it is not containing CD3 or T cell binding domain.Letter " a " after each molecular name indicate specific molecular be in Chinese hamster ovary celI produce and four figures is Mission Number.

Figure 13 depicts the image of Non SDS-PAGE, and display CD3 compares in conjunction with SMIP construct DRA233 so do not tend to fragmentation with DRA234 and SMIP DRA161 (it and DRA209 share identical VH with VL).

Figure 14 depicts the image of reduced form SDS-PAGE, and display CD3 compares in conjunction with SMIP construct DRA233 so do not tend to fragmentation with DRA234 and SMIP DRA161 (it and DRA209 share identical VH with VL).

Figure 15 is display pI modification D RA233 and the DRA234 mainly figure of expressed intact (such as, not carrying out montage) and table.

Figure 16 is the image of the Non CE-SDS of display DRA161 (through purifying).

Figure 17 shows the data of the PK research of DRA234 and DRA233.

The data that the WinNonLin that Figure 18 provides DRA233 and DRA234 come from described in embodiment 14 analyzes.

Figure 19 is the form of the PK estimated value of DRA233 and DRA234.

Figure 20 is the form of the PK parameter that DRA161 and DRA233 (exist and there are not 504 hours points) compares with DRA234.

Figure 21 depicts the result of the target dependent T cell proliferation assay being obtained from the homodimer dual specific peptide molecule using different target CD19.Figure 21 A and 21B respectively illustrates the result of CD4+T cell proliferation as described in Example 10 and CD8+T cell proliferation.

Figure 22 depicts the result of the target dependent T cell proliferation assay being obtained from the heterodimer dual specific peptide molecule using different target CD19.Figure 22 A and 22B respectively illustrates the result of CD8+T cell proliferation as described in Example 10 and CD4+T cell proliferation.

Figure 23 depicts the result being obtained from and using the different polypeptide heterodimers of target CD19 and the redirected T cell cytotoxic assay of homodimer.Figure 23 A shows the result of homologous dimerization dual specific peptide T SC129a, TSC233 and TSC234.Figure 23 B shows the result of heterodimeric dual specific peptide T SC127, TSC227 and TSC228.

Figure 24 depicts the result of the redirected T cell cytotoxic assay being obtained from different dual specific homologous peptide dimer TSC275, TSC277, TSC278 and TSC279 of using target RON.

The result that the T cell that Figure 25 depicts dual specific polypeptide heterodimer and homodimer combines.Figure 25 A and 25B shows bispecific molecule TSC228 (heterodimer) and TSC249 (homodimer) is combined with the dose-dependently of Jurkat cell.

Figure 26 depicts the result of the dimeric target dependent T cell propagation of homologous peptide of target PSMA.Figure 26 A shows in the result with the C4-2B cell of expression target PSMA antigen after TSC249 process.Figure 26 B shows the result not expressing the DU-145 cell of target PSMA antigen after with TSC249 process.Figure 26 C shows the result when T cell separated with TSC249 process.

Figure 27 depicts the result of the redirected T cell cytotoxic assay being obtained from homologous dimerization dual specific peptide T SC194 and TSC249 using target PSMA.

Detailed Description Of The Invention

The invention provides the CD3 Binding peptide of the feature compared with prior art CD3 Binding peptide with improvement.Devise molecule of the present invention to represent the iso-electric point of reduction by the amino acid in modification heavy chain and/or light chain framework region and/or the front hinge area of front hinge to fall low molecular iso-electric point.In one embodiment, modify in the J κ district in Shi VL district and carry out.In some embodiments, modification is amino acid by having positive charge with the aminoacid replacement with neutral charge and/or carries out with the amino acid that the aminoacid replacement with negative charge has a neutral charge.

In some embodiments, the theoretical pI of CD3 Binding peptide of the present invention about 0.5 to 2.5 units lower than AntiCD3 McAb DRA209SMIP (SEQ ID NO:4).For example, the theoretical pI of DRA209 is 9, and the theoretical pI of pI modification D RA219 (SEQ ID NO:6) is 8.4, the theoretical pI of DRA221 (SEQ ID NO:10) is 8.2, the theoretical pI of DRA222 (SEQ IDNO:8) is 7.5, the theoretical pI of DRA223 (SEQ ID NO:12) is 7.2, and the theoretical pI of DRA224 (SEQ ID NO:14) is 6.8.In some embodiments, the experience iso-electric point of CD3 Binding peptide at least 1 pI unit lower than polypeptide SEQ ID NO:4.

Surprisingly, pI variant presents the transformation period of improvement and expresses better than DRA209 in Chinese hamster ovary celI, and such as, see following examples 4, described embodiment describes the method for measuring the transformation period.In addition, compared with having the similar construct of other AntiCD3 McAb binding domain such as such as DRA209 binding domain, the multi specific construct comprising DRA222scFv presents the character (see relevant PCT publication No. WO2012/145714) of improvement.

Chapter title used herein only for organizational goal, and should not be regarded as the theme described by restriction.All documents quoted herein or the part of document, include but not limited to patent, patent application, paper, books and monograph, and the mode quoted all in full is clearly incorporated in this for any object.In one or more term definition in the part of be incorporated to document or document and the conflicting situation of term definition in the application, be as the criterion with the definition occurred in the application.

Unless otherwise noted, otherwise in the present description, any concentration range, percentage range, ratio ranges or integer range are all understood to include any round values in described scope, and when in place, comprise its mark (one of 1/10th and percentage of such as integer).Unless otherwise noted, otherwise as used herein, " about " mean indicated scope, value or structure ± 20%.Should be understood that unless otherwise noted, otherwise " one " and " one " refer to " (kind) or multiple (kind) " in enumerated component as used herein, the term.Alternative word (such as "or") is used to be interpreted as meaning one of sub, both or its any combination.As used herein, term " comprises " and " comprising " uses with synonym.In addition, it should be understood that the application is to comprising the extent of disclosure of polypeptide of component (such as Yu Huo district) described herein and substituent various combination just as individually set forth each polypeptide.Thus, the selection of the specific components of indivedual polypeptide is in the scope of the present disclosure.

As used herein, term " binding domain " or " land " refer to can specific recognition and combine the territory of the protein of the target molecules such as such as antigen, part, acceptor, substrate or inhibitor (such as CD3 or tumor associated antigen, such as RON, CD19, CD37 or PSMA), polypeptide, oligopeptides or peptide, district, part or site.Exemplary combination territory comprises single-chain antibody variable region (such as domain antibodies, sFv, scFv and scFab), receptor extracellular domain and part (such as cytokine, chemokine).In certain embodiments, binding domain comprises the following or is made up of the following: antigen binding site (such as comprising the variable heavy chain sequence and variable light chain sequence or three light chain complementarity determining area (CDR) and three heavy chain CDR that come from the antibody being placed in alternately framework region (FR) (such as optionally comprising the people FR of one or more aminoacid replacement)).The mensuration of the known multiple binding domain for differentiating specific binding particular target of the present disclosure, comprise western blot, ELISA, phage display library screening and interact and analyze.As used herein, CD3 Binding peptide " second binding domain " that can have " CD3 binding domain " and optionally exist.CD3 binding domain can be positioned at amino or C-terminal.In certain embodiments, CD3 binding domain comprises the humanization scFv deriving from mouse monoclonal antibody (such as Cris-7 or HuM291).For example, CD3 binding domain can be made up of the VH district and VL district stemming from mouse monoclonal antibody, wherein uses such as (Gly ₄ser) ₃the connexons such as (SEQ IDNO:76) connexon engage VH district and VL district.In other embodiments, CD3 binding domain in fact by or be made up of the humanization scFv stemming from mouse monoclonal antibody.In some embodiments, CD3 binding domain of the present invention comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 of muroid Cris-7 antibody or HuM291.

If binding domain is to be equal to or greater than 10 ⁵m ^-1avidity or K _a(that is, the interactional equilibrium association constant of particular combination, unit is 1/M) does not significantly combine other component existed in test sample in conjunction with target, then its " specific binding " target.Binding domain can be categorized as " high-affinity " binding domain and " low-affinity " binding domain." high-affinity " binding domain refers to have at least 10 ⁷m ^-1, at least 10 ⁸m ^-1, at least 10 ⁹m ^-1, at least 10 ¹⁰m ^-1, at least 10 ¹¹m ^-1, at least 10 ¹²m ^-1or at least 10 ¹³m ^-1k _athose binding domain." low-affinity " binding domain refers to have maximum 10 ⁷m ^-1, maximum 10 ⁶m ^-1, maximum 10 ⁵m ^-1k _athose binding domain.Or avidity can be defined as the interactional equilibrium dissociation constant (K of particular combination _d), unit is M (such as 10 ^-5m is to 10 ^-13m).Avidity according to binding domain polypeptide of the present disclosure and single chain polypeptide can use routine techniques easily to measure (see (1949) Ann.N.Y.Acad.Sci.51:660 such as such as Scatchard; With U.S. Patent number 5,283,173,5,468,614 or equivalent).

Known in this area " CD3 " be six chains multiprotein complex (see such as Abbas and Lichtman, 2003; Janeway etc., the 172nd and 178 pages, 1999), described six chains are subunits of tcr complex.In Mammals, the CD3 subunit of T-cell receptors mixture is CD3 γ chain, CD3 δ chain, two CD3 ε chains and CD3 ζ chain homodimer.CD3 γ, CD3 δ and CD3 ε chain are the height correlation cell surface proteins of the immunoglobulin superfamily containing single immunoglobulin domain.The cross-film zone negative electricity of CD3 γ, CD3 δ and CD3 ε chain, this is the feature allowing the φt cell receptor chain of these chains and positively charged to associate.The single consensus motif of afterbody separately containing the activation motifs be called based on immunity receptor tyrosine or ITAM in the cell of CD3 γ, CD3 δ and CD3 ε chain, and each CD3 ζ chain has three.Believe that ITAM is important for the intracellular signaling ability of TCR mixture.CD3 as used in this disclosure can come from different animal species, comprises people, monkey, mouse, rat or other Mammals.

As used herein, " guard replacement " and be acknowledged as another amino acid that an aminoacid replacement has similar quality in the art.Exemplary conservative replace be in the art well-known (see such as WO97/09433 disclosed in 13 days March in 1997, the 10th page; Lehninger, Biochemistry, the second edition; Worth Publishers, Inc.NY:NY (1975), 71-77 page; Lewin, Genes IV, Oxford University Press, NY and CellPress, Cambridge, MA (1990), the 8th page).In certain embodiments, conservative replacement comprises leucine and replaces Serine.

As used herein, term " derivative " refers to when presence or absence enzyme by chemistry or biological means, such as, formed the modification of the one or more amino-acid residues to peptide by glycosylation, alkylation, acidylate, ester formation or acid amides.

As used herein, the source of specifying the polypeptide of polypeptide or protein or aminoacid sequence to refer to polypeptide " is stemmed from ".In one embodiment, stem from the polypeptide of particular sequence or aminoacid sequence have in fact with this sequence or the same aminoacid sequence of its part, wherein said part by least 10-20 amino acid, at least 20-30 amino acid or at least 30-50 amino acid or at least 50-150 amino acid form; Or those skilled in the art otherwise can differentiate it, because have its source in the sequence.In one embodiment, humanization or chimeric binding domain stem from VH and/or VL of the antibody produced by another animal.For example, humanization binding domain or chimeric binding domain may stem from VH and/or the VL district of rodent antibody.For example, can by modifying by method as known in the art the humanization that rodent antibody is carried out in framework region.

The polypeptide stemming from another polypeptide may have one or more sudden change relative to starting polypeptide, and such as one or more amino-acid residue replaces through another amino-acid residue or it has the insertion of one or more amino-acid residue or disappearance.Polypeptide may comprise the aminoacid sequence that non-natural exists.Described variant and starting polypeptide must have the sequence iden or the similarity that are less than 100%.In one embodiment, the aminoacid sequence of variant and the aminoacid sequence of starting polypeptide will have about 60% to the amino acid sequence identity or the similarity that are less than 100%.In another embodiment, the aminoacid sequence of variant and the aminoacid sequence of starting polypeptide will have about 75% to being less than 100%, about 80% to being less than 100%, about 85% to being less than 100%, about 90% to being less than 100%, about 95% to the amino acid sequence identity or the similarity that are less than 100%.

As used herein, unless specified otherwise herein, otherwise the position of amino-acid residue in the variable region of immunoglobulin molecules is according to Kabat numbering regulation (Kabat, Sequences ofProteins of Immunological Interest, 5th edition, Bethesda, MD:PublicHealth Service, National Institutes of Health (1991)) be numbered, and the position of amino-acid residue in the constant region of immunoglobulin molecules is numbered according to EU nomenclature (Ward etc., 1995Therap.Immunol.2:77-94).

As used herein, term " dimer " refers to and (comprises covalent linkage, such as disulfide linkage by the intramolecular force via one or more forms; Interact with other, such as electrostatic interaction, salt bridge, hydrogen bonding and hydrophobic interaction) two subunits composition of associating each other and at felicity condition (such as, in physiological conditions, at applicable expression, purifying and/or store in the aqueous solution of recombinant protein, or under the condition for non denatured and/or non-reduced electrophoresis) under stable biological entities." heterodimer " or " heterodimeric body protein " refers to the dimer formed by two different polypeptide as used herein.Heterodimer does not comprise the antibody formed by four polypeptide (that is, two light chains and two heavy chains).In one embodiment of the invention, the Interceptor platform comprising the Emergent in heterodimeric territory is used to produce heterodimer." homodimer " or " heterodimeric protein " refers to the dimer formed by two same polypeptide as used herein.In certain embodiments of the invention, SMIP, PIMS or Scorpion platform of Emergent is used to produce homodimer.

As used herein, " hinge area " or " hinge " refers to and stems from following polypeptide: the Yu Jian district of (a) transmembrane protein (such as I type transmembrane protein); Or the district of stem (stalk region) of (b) II type C-lectin.For example, hinge area can stem from the Yu Jian district of immunoglobulin superfamily member; Suitable hinge area in this particular category comprises (i) immunoglobulin hinge region (for example, being made up of upper zone and core area) or its functional variant, comprises the immunoglobulin (Ig) hinge of wild-type and change; (ii) region (or its functional variant) in immunoglobulin (Ig) V sample or immunoglobulin (Ig) C spline structure territory is connected.

" wild-type immunoglobulin hinge area " refers to being inserted between CH1 and CH2 territory of finding in heavy chain of antibody and connects CH1 and CH2 territory (for IgG, IgA and IgD) or to be inserted between CH1 and CH3 territory and the naturally occurring top of connection CH1 and CH3 territory (for IgE and IgM) and middle hinge aminoacid sequence.In certain embodiments, wild-type immunoglobulin hinge legion sequence is people, and can comprise human IgG hinge area.

" the wild-type immunoglobulin hinge area of change " or " immunoglobulin hinge region of change " refer to that (a) has maximum 30% amino acid change (such as, maximum 25%, 20%, 15%, 10% or 5% aminoacid replacement or disappearance) wild-type immunoglobulin hinge area, or in (b) wild-type immunoglobulin hinge area, length is about 5 amino acid (such as about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid) to maximum about 120 amino acid (such as, length is about 10 to about 40 amino acid or about 15 to about 30 amino acid or about 15 to about 20 amino acid or about 20 to about 25 amino acid), there is about 30% amino acid change (such as, at most about 25% at most, 20%, 15%, 10%, 5%, 4%, 3%, 2% or 1% aminoacid replacement or disappearance or its combination) and have as PCT announces the part of IgG core hinge area disclosed in WO2011/090762 and WO2011/090754.

As used herein, term " humanization " refers to by using genetic engineering technique to make antibody or immunoglobulin-binding proteins have more weak immunogenicity with the polypeptide stemming from non-human species (such as mouse or rat) to people, still retains the process of the antigen-binding matter of original antibodies simultaneously.In some embodiments, by the binding domain humanization of antibody or immunoglobulin-binding proteins and polypeptide (such as, light chain and variable region of heavy chain, Fab, scFv).The technology and modification thereof that are called that CDR transplants can be used inhuman binding domain humanization (Jones etc., Nature 321:522 (1986)), described modification comprises " remodeling " (Verhoeyen etc., 1988Science239:1534-1536; Riechmann etc., 1988Nature 332:323-337; Tempest etc., Bio/Technol 19919:266-271), " high chimeric " (Queen etc., 1989Proc Natl AcadSci USA 86:10029-10033; Co etc., 1991Proc Natl Acad Sci USA88:2869-2873; Co etc., 1992J Immunol 148:1149-1154) and " facing " (Mark etc., " Derivation of therapeutically active humanized and veneeredanti-CD18antibodies. " Metcalf BW, Dalton BJ compiles, Cellular adhesion:molecular definition to therapeutic potential.New York:Plenum Press, 1994:291-312).If stem from inhuman source, then other district of antibody or immunoglobulin-binding proteins and polypeptide, such as hinge area and constant region domain, also can carry out humanization.

" constant region for immunoglobulin " or " constant region " is the term defined herein, and it refers to the peptide or the peptide sequence that correspond to or stem from part or all of one or more constant region domain.In one embodiment, constant region is containing all constant region domain of active antibody.In one embodiment, constant region comprises IgG CH2 and CH3 territory, such as IgG1CH2 and CH3 territory.In some embodiments, constant region does not comprise CH1 territory.In certain embodiments, the constant region domain forming constant sub-region is people.In some embodiments, the constant region domain of fusion rotein of the present disclosure lacks or has the cytotoxicity (ADCC) of few antibody dependent cellular mediation and complement activation and supply the effector function of dependent cellular cytotoxicity (CDC), and the ability retained in conjunction with some Fc acceptors (such as FcRn, i.e. neonatal Fc receptor) and retain the relatively long transformation period in vivo.

In other change programme, fusion rotein of the present disclosure comprises the constant domain retaining one of ADCC and CDC or both described effector functions.In certain embodiments, binding domain of the present disclosure and human IgG constant region (such as IgG1) merge, and wherein said constant region has the amino acid of one or more following sudden change: the Methionin (K322) on the glycine (G237) on the leucine (L234) on position 234, the leucine (L235) on position 235, position 237, the glutamate (E318) on position 318, the Methionin (K320) on position 320, position 322 or its any combination (being numbered according to EU).For example, any one or more in these amino acid can be become L-Ala.In another embodiment, L234, L235, G237, E318, K320 and K322 in IgG Fc territory (such as IgG1) (according to EU numbering) are mutated into such as that L-Ala is (namely separately, be respectively L234A, L235A, G237A, E318A, K320A and K322A) and the N297A sudden change (that is, eliminating the glycosylation in CH2 territory in fact) that optionally exists.

As used herein, term " little module formula immune drug albumen " or " SMIP " are used in reference to generally as protein scaffolds disclosed in such as U.S. Patent Application Publication No. 2003/0133939,2003/0118592 and 2005/0136049, and the mode that described document is quoted in full is incorporated herein.In this article, embodiment and disclosure in full described in " SMIP molecule " be interpreted as comprising SMIP support, such as comprise the associated proteins of the first binding domain, hinge area and constant region for immunoglobulin according to the order from amino to C-terminal." CD3 specificity SMIP molecule " is interpreted as the CD3 associated proteins comprising SMIP support.

As used herein, term " PIMS " is used in reference to generally as protein scaffolds disclosed in such as U.S. Patent Application Publication No. 2009/0148447, and the mode that described document is quoted in full is incorporated herein.In this article, embodiment and disclosure in full described in " PIMS molecule " be interpreted as comprising PIMS support, such as comprise the associated proteins of constant region for immunoglobulin, hinge area and the first binding domain according to the order from amino to C-terminal." CD3 specificity PIMS molecule " is interpreted as the CD3 associated proteins comprising PIMS support.

" Fc district " or " Fc territory " refers to the peptide sequence corresponding to or stem from the antibody of source the part be responsible in conjunction with the antibody receptor on cell and the C1q component on complement.Fc represents " crystallizable fragment ", namely easily forms the antibody fragment of protein crystal.The particular protein fragment described by proteolytic digestion at first can define the overall universal architecture of immunoglobulin (Ig).Define as initial in document, the heavy chain hinge region that Fc fragment is connected by disulphide, CH2 and CH3 territory form.But this term has been applied to by CH3, CH2 and the strand formed at least partially being enough to the dimeric hinge that the chain formation disulphide such with second is connected recently.About the review of immunoglobulin structure and function, see Putnam, The PlasmaProteins, V volume (Academic Press, Inc., 1987), 49-140 page; And Padlan, Mol.Immunol.31:169-217,1994.As used herein, term Fc comprises the variant of naturally occurring sequence.

As used herein, the pH value that " iso-electric point " or pI are net charges when being zero.

As used herein, term " Interceptor " is used in reference to generally as PCT announces monospecific disclosed in WO2011/090762 and WO2011/090754 or polyspecific heterodimeric protein scaffolds.Interceptor molecule described herein is interpreted as the CD3 associated proteins comprising two non-same polypeptide chains, and each polypeptide chain comprises immunoglobulin (Ig) heterodimeric territory.Immunoglobulin (Ig) heterodimeric territory, interface is different.In one embodiment, immunoglobulin (Ig) heterodimeric territory comprises CH1 territory or derivatives thereof.In another embodiment, immunoglobulin (Ig) heterodimeric territory comprises CL territory or derivatives thereof.In one embodiment, CL territory is C κ or C λ isotype or derivatives thereof.

As used herein, " district of stem " of II type C-lectin refers in the extracellular of II type C-lectin and is positioned at C-type lectin-like domain (CTLD; Such as, be similar to the CTLD of Natural Killer Cell Receptors) and membrane-spanning domain between part.For example, in people CD94 molecule (GenBank accession number AAC50291.1, PRI, November 30 nineteen ninety-five), extracellular corresponds to amino-acid residue 34-179, and CTLD corresponds to amino-acid residue 61-176.Therefore, the district of stem of people CD94 molecule comprises amino-acid residue 34-60, it is found that it is between film and CTLD (see Boyington etc., Immunity 10:75,1999; About the description in other district of stem, also see Beavil etc., Proc.Nat'l.Acad.Sci.USA89:753,1992; With Figdor etc., Nature Rev.Immunol.2:77,2002).These II type C-lectins can also district of stem with between cross-film district or CTLD, there are 6 to 10 and engage amino acid.In another embodiment, 233 amino acid whose people NKG2A albumen (GenBank accession number P26715.1, PRI, on June 15th, 2010) have the cross-film district within the scope of amino acid 71-93 and the extracellular within the scope of amino acid 94-233.CTLD is made up of amino acid/11 19-231, and district of stem comprises amino acid 99-116, and both sides are connected with two amino acid by five.Between other II type C-lectin and its extracellular ligand binding domain, territory or district of stem, be well known in the art (about human CD 23, CD69, CD72, NKG2A and NKG2D and its description, respectively see such as GenBank accession number NP_001993.2 with CTLD; AAH07037.1, PRI, on July 15th, 2006; NP_001773.1, PRI, on June 20th, 2010; AAL65234.1, PRI, on January 17th, 2002; And CAA04925.1, PRI, on November 14th, 2006).

As used herein, " the Yu Jian district " of transmembrane protein (such as I type transmembrane protein) refers to the part between two adjacent domains in the extracellular of transmembrane protein.The region that the example in Yu Jian district comprises the adjacent Ig territory connecting immunoglobulin superfamily member (such as, comes from the immunoglobulin hinge region of IgG, IgA, IgD or IgE; Connect the IgV territory of CD2 and the region in IgC2 territory; Or connect the IgV territory of CD80 or CD86 and the region in IgC territory).Another example in Yu Jian district connects the non-Ig territory of CD22 (one is in conjunction with I type sialic Ig sample lectin) and the region in IgC2 territory.

" stem from " II type C-district of lectin stem or " stemming from " transmembrane protein Yu Jian district (such as, immunoglobulin hinge region) peptide zone refer to about 5 to about 150 aminoacid sequences, its overall or part comprises region sequence between district of (i) wild-type stem or territory; (ii) fragment of region sequence between district of wild-type stem or territory; (iii) there is with (i) or (ii) polypeptide of at least 80%, 85%, 90% or 95% amino acid sequence identity; (iv) wherein one, two, three, four or five amino acid there is disappearance, insertion, replace or (i) or (ii) of its any combination, such as one or more change replaces, or one or more sudden change only comprises a disappearance.In some embodiments, with wild-type stem region sequence, such as stem from NKG2A, NKG2D, CD23, CD64, CD72 or CD94 about 8 compare to about 20, about 10 to about 25 or about 15 to about 25 those sequences amino acid whose, and stem's district's derivative has more resistance to proteolytic cleavage.

As used herein, between two adjacent areas that term " joint amino acid " or " joint amino-acid residue " refer to polypeptide or structural domain, such as between hinge and adjacent immunoglobulin (Ig) constant sub-region, or between hinge and adjacent binding domain, or connect one or more (the such as about 2-10) amino-acid residue between the peptide connexon of two immunoglobulin variable domain and adjacent immunoglobulin variable domain.Engage amino acid to be produced (such as, by the amino-acid residue using restriction enzyme sites to produce during building the nucleic acid molecule of coded polypeptide) by polypeptide construct design.Joint amino acid between binding domain and hinge area is referred to herein as front hinge area, such as, can produce by interpolation restriction site to coding nucleotide sequence.In one embodiment, front hinge area is made up of about 1,2,3,4,5,6,7,8,9,10 or 15 amino acid.The front hinge legion sequence of DRA209 and DRA161 is not a part for muroid Cris-7 or human normal immunoglobulin germline sequence.The front hinge area of DRA209 and DRA161 is aminoacid sequence RRT, and for DRA219, DRA221, DRA222, DRA223, DRA224, DRA225, DRA228, DRA229, DRA233, DRA234, TSC249, TSC250, TSC251, TSC252, TSC295, TSC296, TSC301 and TSC302, it is SSS.Binding molecule of the present invention may comprise or may not comprise front hinge area.

As used herein, phrase " connexon between CH3 and CH1 or CL " refers to one or more (the such as about 2-12) amino-acid residue between the C-terminal in CH3 territory (such as wild-type CH3 or saltant type CH3) and the N-terminal of CH1 territory or CL territory (such as Ck).

As used herein, term " patient in need " refers to that risky suffer from or just suffering from can with the patient of provided CD3 associated proteins or polypeptide or the treatment of its composition or the disease improved, illness or symptom herein.

As used herein, term " peptide connexon " refers to that connecting variable region of heavy chain with variable region of light chain provides the spacer function compatible with the interaction of two sub-binding domain to retain as comprising the aminoacid sequence of identical light chain with the specific binding avidity of the identical target molecule of the antibody of variable region of heavy chain to make gained polypeptide.In certain embodiments, connexon by 5 to about 35 amino acid, such as about 15 to about 25 Amino acid profiles.

As used herein, term " pharmaceutically acceptable " refers to that molecular entity and composition can not produce supersensitivity or the reaction of other serious harm in most of experimenter when using approach well known in the art to use.Administration's approval of federal or state government or the molecular entity of for animal and or rather confession people of registering in American Pharmacopeia or other pharmacopeia that It is generally accepted and composition be considered to be " pharmaceutically acceptable ".

As used herein, term " promotor " refers to and relates in conjunction with RNA polymerase with the region of DNA territory of initiation transcription.

As used herein, term " nucleic acid ", " nucleic acid molecule " or " polynucleotide " refer in strand or the deoxyribonucleotide of double chain form or ribonucleotide and its polymkeric substance.Unless there are concrete restriction, otherwise this term is contained and is had similar binding properties with reference nucleic acid and with the nucleic acid containing natural nucleus glycoside acid-like substance of the mode metabolism being similar to naturally occurring Nucleotide.Unless otherwise noted, otherwise the specific nucleic acid sequence sequence that also implies its variant modifying (such as degenerate codon replacement) through conservative and complementary sequence and clearly indicate.Specifically, degenerate codon replacement can realize (Batzer etc. (1991) Nucleic Acid Res.19:5081 by the 3rd position producing one or more selected (or all) codons through the sequence of mixing base and/or deoxyinosine residue replacement; Ohtsuka etc. (1985) J.Biol.Chem.260:2605-2608; Cassol etc. (1992); Rossolini etc. (1994) Mol.Cell.Probes8:91-98).Term nucleic acid and gene, cDNA and the mRNA by genes encoding are used interchangeably.As used herein, term " nucleic acid ", " nucleic acid molecule " or " polynucleotide " are intended to comprise DNA molecular (such as cDNA or genomic dna), RNA molecule (such as mRNA), DNA or the RNA analogue using nucleotide analog to produce and its derivative, fragment and homologue.

Term " expression " refers to by the biosynthesizing of the product of nucleic acid encoding.For example, when encoding the nucleic acid segment of related polypeptide, expressing to relate to nucleic acid segment to transcribe in mRNA and by mRNA and translating into one or more polypeptide.

Term " expression unit " and " expression cassette " are used interchangeably in this article, and presentation code related polypeptide and can in host cell the nucleic acid segment of express nucleic acid section.Express unit and typically comprise transcripting promoter, the coding open reading frame of related polypeptide and transcription terminator, all in exercisable configuration.Except transcripting promoter and terminator, express unit and may further include other nucleic acid segment, such as enhanser or polyadenylation signal.

As used herein, term " expression vector " refers to linear or circular nucleic acid molecule, and it comprises one or more expression unit.Except one or more expression unit, expression vector can also comprise other nucleic acid segment, such as one or more replication orgin or one or more selectable marker thing.Expression vector generally stems from plasmid or viral DNA, maybe can containing both elements.

As used herein, Scorpion is the term being used in reference to multi-specific binding protein support.For example, PCT application publication No. WO 2007/146968, U.S. Patent Application Publication No. 2006/0051844, PCT application publication No. WO 2010/040105, PCT application publication No. WO2010/003108 and U.S. Patent number 7,166, disclose multi-specific binding protein and polypeptide in 707.Scorpion polypeptide comprises two basic change territory (described structural domain can be designed for the identical or different target of specific binding), two connexons and an immunoglobulin (Ig) constant sub-region.The connexon for Scorpion molecule is described in PCT application publication No. WO2010/003108.In some embodiments, connexon sequence is an about 2-45 amino acid or 2-38 amino acid or 5-45 amino acid.In some embodiments, connexon is antibody hinge region (such as coming from IgG) or district of C-lectin stem.Scorpion albumen comprises two same homologous dimerization albumen by the Scorpion polypeptide of disulphide bonding.

As used herein, term " sequence iden " refers to the relation between two or more polynucleotide sequences or between two or more peptide sequences.When the position in a sequence is occupied by the identical nucleic acid base on the correspondence position of comparative sequences or amino-acid residue, described sequence is called on that position " same "." sequence iden " per-cent is that the positional number by determining to exist in two sequences same nucleic acid base or amino-acid residue calculates to produce " same " positional number.Then " same " positional number is multiplied by 100, to produce " sequence iden " per-cent divided by the total positional number in comparison window." sequence iden " per-cent is determined by the sequence comparing two best comparisons in comparison window.The length of the comparison window of nucleotide sequence can be such as at least 20,30,40,50,60,70,80,90,100,110,120,130,140,150,160,170,180,190,200,300,400,500,600,700,800,900 or 1000 or more nucleic acid.The length of the comparison window of peptide sequence can be such as at least 20,30,40,50,60,70,80,90,100,110,120,130,140,150,160,170,180,190,200,300 or more amino acid.In order to best aligned sequences is for comparing, the polynucleotide in comparison window or peptide sequence part can comprise the interpolation or disappearance that are called room, and reference sequences keeps constant.Even if best comparison also can produce the comparison of maximum possibility " same " positional number between reference sequences and comparative sequences when being and having vacant position." sequence iden " per-cent between two sequences can be determined by service routine version " BLAST 2Sequences ", this program version can derive from NCBI's (by the end of on September 1st, 2004), this program is incorporated with program BLASTN (for nucleotide sequence comparison) and BLASTP (comparing for peptide sequence), these two programs are the algorithms based on Karlin and Altschul (Proc.Natl.Acad.Sci.USA90 (12): 5873-5877,1993).When utilizing " BLAST 2Sequences ", parameter (namely, default parameter by the end of on September 1st, 2004) may be used for word length (3), open gap penalty (11), extend gap penalty (1), room decline (50), expected value (10) and other desired parameters any, include but not limited to matrix option.If two Nucleotide or aminoacid sequence relative to each other have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence iden, then these two sequences are regarded as having " substantially similar sequence iden " or " basic sequence iden ".

As used herein, " polypeptide " or " polypeptide chain " is covalently bound amino acid whose single linear and continuous arrangement.It does not comprise in a non-linear manner, two polypeptide chains such as linking together via interchain disulfide bond (such as, light chain be connected via disulfide linkage with heavy chain half immunoglobulin molecule).Polypeptide can have or be formed one or more intrachain disulfide bond.About polypeptide as described in this article, mention the posttranslational modification that the amino-acid residue corresponding to those amino-acid residues illustrated by SEQ ID NO comprises described residue.

" protein " is the macromole comprising one or more polypeptide chain.Protein can also comprise non-peptide composition, such as carbohydrate group.Carbohydrate and other non-peptide substituting group can add protein to by the protedogenous cell of product, and will change with cell type.Protein is define according to its amino acid backbone structures in this article; Generally the substituting groups such as such as carbohydrate group are not described, but still may exist.

Term " N-terminal " (N-terminal) and " C-terminal " (C-terminal) are in this article for representing the position in polypeptide.When context allows, these terms use to represent contiguous or relative position with reference to particular sequence or polypeptide portion.For example, some sequence being positioned at C-terminal relative to reference sequences in polypeptide is positioned near the C-terminal of reference sequences, but may not on the C-terminal of complete polypeptide.

" φt cell receptor " (TCR) is the molecule found on the surface in T cell, is generally responsible for identifying the antigen be combined with MHC (MHC) molecule together with CD3.In most of T cell, the heterodimer that it is connected with the disulphide of β chain by alterable height α forms.In other T cell, express the replacement acceptor be made up of variable γ and δ chain.Each chain of TCR is the member of immunoglobulin superfamily, and have a N-terminal immunoglobulin variable domain, immunoglobulin (Ig) constant domain, cross-film district and C-terminal short kytoplasm tail (see Abbas and Lichtman, Cellular and Molecular Immunology (the 5th edition), editor: Saunders, Philadelphia, 2003; Janeway etc., Immunobiology:TheImmune System in Health and Disease, the 4th edition, Current BiologyPublications, the 148th, 149 and 172 page, 1999).TCR as used in this disclosure can come from various animal species, comprises people, mouse, rat or other Mammals.

" TCR mixture " refers to and to be associated the mixture formed by CD3 chain and other TCR chain as used herein.For example, TCR mixture can be made up of the homodimer of CD3 γ chain, CD3 δ chain, two CD3 ε chains, CD3 ζ chain, TCR α chain and TCR β chain.Or TCR mixture can be made up of the homodimer of CD3 γ chain, CD3 δ chain, two CD3 ε chains, CD3 ζ chain, TCR γ chain and TCR δ chain.

As used herein " component of TCR mixture " refer to TCR chain (such as TCR α, TCR β, TCR γ or TCR δ), CD3 chain (such as CD3 γ, CD3 δ, CD3 ε or CD3 ζ) or by two or more TCR chains or CD3 chain formation mixture (mixture of such as TCR α and TCR β, the mixture of TCR γ and TCR δ, the mixture of CD3 ε and CD3 δ, the mixture of CD3 γ and CD3 ε, or TCR α, TCR β, CD3 γ, CD3 δ and two CD3 ε chains sub-TCR mixture).

" cytotoxicity of antibody dependent cellular mediation " and " ADCC " refer to cell-mediated process as used herein, wherein express the nonspecific cytotoxic cells of Fc γ R (such as, monocyte, such as natural killer (NK) cell and scavenger cell) identify binding antibody on target cell (maybe can in conjunction with the other oroteins of Fc γ R) and cause target cell lysis subsequently.In principle, any effector cell with activation Fc γ R may be triggered and mediate ADCC.Mediation ADCC primary cell be NK cell, it only expresses Fc γ RIII, and monocyte depend on they active state, location or differentiation can express Fc γ RI, Fc γ RII and Fc γ RIII.About the review that the Fc γ R on hematopoietic cell expresses, see such as Ravetch etc., 1991, Annu.Rev.Immunol., 9:457-92.

Term as used when mentioning polypeptide or protein herein " has ADCC activity " and means polypeptide or protein (such as, comprise the immunoglobulin hinge region and immunoglobulin (Ig) constant sub-region with CH2 and CH3 territory, such as stem from polypeptide or the protein (such as IgG1) of IgG) cytotoxicity (ADCC) that the cytolytic Fc acceptor (such as Fc γ RIII) combined on the cytolytic immune effector cell (such as NK cell) of expressing Fc acceptor carry out the mediation of mediate antibody dependent cell can be passed through.

" CDC " and " CDC " refers to component (" complement ") in normal serum and antibody or represents the process of the dissolving of the target cell of expressing target antigen together with other C1q complement associated proteins of combining with target antigen as used herein.Complement acts synergistically by one group and ordered arrangement forms with the serum protein playing its effect.

" classical complement pathway " and " classical complement system " is synonym as used herein, the term, and refers to the particular path of complement activation.Classical path needs antigen-antibody complex so that initial sum participates in the activation of nine kinds of chief protein component (being appointed as C1 to C9) in an orderly manner.For several steps in reactivation process, product is the enzyme of energy catalysis subsequent step.The relatively little start signal of this grade of coupling increases and has activated a large amount of complement.

Polypeptide or protein as described in term as used about polypeptide or protein herein " has CDC activity " and means are (such as, comprise the immunoglobulin hinge region and immunoglobulin (Ig) constant sub-region with CH2 and CH3 territory, such as stem from polypeptide or the protein (such as IgG1) of IgG) can by carrying out mediate complement dependent cellular cytotoxicity (CDC) in conjunction with C1q complement proteins and the classical complement system of activation.

" redirected T cell cytotoxicity " and " RTCC " refer to the process that T cell mediates as used herein, wherein using can specific binding cytotoxic T cell and target cell, and cytotoxic T cell is raised target cell by the polyspecific albumen caused whereby for the target dependent cellular cytotoxicity t cell responses of target cell.

As used herein, term " treatment (treatment) ", " treatment (treating) " or " alleviation " refer to therapeutic treatment or precaution/prophylactic treatment.If at least one disease symptoms connect in subject individuality is improved or treats the deterioration of the PD that can postpone in individuality or prevents other relative disease from showing effect, then treatment is for curative.

As used herein, term " conversion ", " transfection " and " transduction " refer to and are transferred in cell by nucleic acid (such as nucleotide polymer).As used herein, term " genetic transformation " refers to DNA, and especially recombinant DNA shifts and is incorporated in cell.The nucleic acid of transfer can be incorporated in cell via expression vector.

As used herein, term " variant " refers to and is different from reference nucleic acid or polypeptide, but retains nucleic acid or the polypeptide of its essential property.In general, variant overall is similar to closely reference nucleic acid or polypeptide and is same with reference nucleic acid or polypeptide in many regions.For example, compared with active part or total length reference nucleic acid or polypeptide, variant can represent at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence iden.

Term " variable region of light chain " (also referred to as " light-chain variable domain " or " VL ") and " variable region of heavy chain " (also referred to as " heavy chain variable domain " or " VH ") refer to the variable land coming from light chain of antibody and heavy chain respectively.Variable land is by being called that the discrete well-defined subprovince of " complementarity-determining region " (CDR) and " framework region " (FR) forms.In one embodiment, FR is by humanization.Term " CL " refers to " immunoglobulin light chain constant district " or " constant region of light chain ", that is, come from the constant region of light chain of antibody.Term " CH " refers to " immunoglobulin heavy chain constant region " or " CH ", depend on antibody isotype, it can also be further divided into CH1, CH2 and CH3 (IgA, IgD, IgG) or CH1, CH2, CH3 and CH4 territory (IgE, IgM)." Fab " (Fab) is the part of conjugated antigen in antibody and comprises the variable region and CH1 territory that are connected with light chain via intrachain disulfide bond in heavy chain.

The disclosure particularly provides and comprises binding domain, exactly, comprises the peptide and protein of first binding domain of specific binding CD3.It is one or more that the peptide and protein comprising binding domain of the present disclosure can comprise in the following: immunoglobulin (Ig) constant sub-region, connection peptides, hinge area, immunoglobulin (Ig) heterodimeric territory, immunoglobulin (Ig) dimerization territory, one or more joint amino acid, label etc.These components of disclosed peptide and protein are described in further detail herein.

In addition, CD3 Binding peptide disclosed herein and protein can in multiple multi-form in the antibody of any one or the form (such as, fusion rotein can be SMIP albumen, PIMS albumen, Scorpion albumen or Interceptor protein form) of fusion rotein.

In some embodiments, CD3 Binding peptide comprises the second binding domain.In some embodiments, CD3 Binding peptide comprises CD3 binding domain, hinge area, constant region and the second binding domain from amino to C-terminal.In other embodiments, CD3 Binding peptide comprises the second binding domain, hinge area, constant region and CD3 binding domain from amino to C-terminal.

CD3 associated proteins according to the present invention generally comprises (a), and at least one comprises the CD3 Binding peptide chain of CD3 binding domain as described herein.In some change programme, CD3 Binding peptide also comprises hinge area and (c) constant region for immunoglobulin (such as SMIP polypeptide) that (b) C-terminal is connected to CD3 binding domain.In other change programme, CD3 Binding peptide also comprises the second hinge area that (d) C-terminal is connected to immunoglobulin (Ig) constant sub-region, and (e) C-terminal is connected to second binding domain (such as Scorpion polypeptide) of the second hinge area.In some embodiments, CD3 Binding peptide comprises the CD3 binding domain with framework region, front hinge area and hinge area.

In some embodiments, CD3 Binding peptide comprises three or more amino acid modified compared with SEQ IDNO:42, for example, described modification is with the amino acid of neutral amino acids replacement positively charged and/or with electronegative aminoacid replacement neutral amino acids, such as, in framework region and/or front hinge area.In some embodiments, by replace with neutral amino acids positively charged amino acid and/or with electronegative aminoacid replacement neutral amino acids in the framework region of VH and/or VL at least 2, at least 3, at least 4,3-5 or 3-10 amino acid modifies.

In some embodiments of the present invention, CD3 Binding peptide comprises the modification of the pI that such as can reduce front hinge area or whole polypeptide in front hinge area.Front hinge area is in the joint between binding domain and hinge area.For example, three amino acid whose front hinge areas of sequence RRT can be had to reduce iso-electric point with sequence SSS or SST displacement.In some embodiments, front hinge area has the iso-electric point of reduction compared with having the CD3 Binding peptide of the front hinge area of RRT.

In one embodiment, in order to reduce immunogenic risk, by on identical in ubiquitous in human germ-line system IgG sequence or human germ-line system sequence or contiguous position, the aminoacid replacement such as contained in the human germ-line system IgG sequence of correspondence is in sequence.In some embodiments, described human germ-line system IgG sequence comprises SEQ ID NO:43 or 44.

In some embodiments, compared with Cris-7VL J κ (Jk) district mouse sequence LQIT (SEQID NO:252), at least one is amino acid modified in the Jk district in VL district.In some embodiments, Jk district comprises aminoacid sequence VEIK (SEQ ID NO:253), such as, replace LQIT (SEQ ID NO:252).

In some embodiments, CD3 Binding peptide comprises hinge area and constant region, for example, CD3 Binding peptide can comprise the hinge area that (a) N-terminal is connected to CD3 binding domain, (b) N-terminal is connected to the constant region for immunoglobulin (such as, PIMS polypeptide) of hinge area.In some embodiments, CD3 Binding peptide comprises CD3 binding domain, hinge area and constant region from amino to C-terminal.In some embodiments, CD3 Binding peptide comprises constant region, hinge area and CD3 binding domain from amino to C-terminal.

Typically, the CD3 Binding peptide (SMIP, Scorpion or PIMS) of above-mentioned form can carry out homologous dimerization, typically via disulfide linkage, via constant region for immunoglobulin and/or hinge area (such as, via the constant region for immunoglobulin and the IgG hinge area that comprise IgG CH2 and CH3 territory).Thus, of the present invention in some, two same CD3 Binding peptides carry out homologous dimerization, to form dimerization CD3 associated proteins.

In other embodiments, CD3 Binding peptide also comprises the heterodimeric territory can carrying out heterodimeric from the different heterodimeric territory in the second non-same polypeptide chain.In some change programme, the second polypeptide chain for heterodimeric comprises the second binding domain.Therefore, of the present invention in some, two non-same polypeptide chains (one comprise CD3 binding domain and second optionally comprise the second binding domain) dimerization is to form the heterodimeric protein with 1 to 4 binding domain.In some embodiments, CD3 Binding peptide the second binding domain of comprising CD3 binding domain, hinge area, constant region, heterodimeric territory and optionally exist from amino to C-terminal.

In some embodiments, the second binding domain binding target molecule or with its interaction, and CD3 Binding peptide inducing T cell cytotoxicity.This can be non-dimeric body, heterodimer or homodimer form.In some embodiments, the second binding domain in conjunction with tumor associated antigen or with its interaction.In some embodiments, CD3 Binding peptide dissolves tumour cell at tumor vicinity inducing T cell and/or induces polyclone T cell activation and amplification.

The polypeptide comprising AntiCD3 McAb binding domain of the present invention can be or comprise antibody or include the reservation function antibody fragment of binding specificity or the antibody derivatives of fragment derivatives.In some embodiments, the present invention includes the fusion rotein containing variable heavy chain territory and/or light chain territory and other polypeptide.

In some embodiments of the present invention, CD3 Binding peptide contain include be selected from SEQ ID NO:28,30,32, the VH region sequence of 38 and 40 or the CD3 binding domain of VL region sequence.In some embodiments, CD3 binding domain comprise be selected from SEQ ID NO:22,24,26,28, the VH district of 30 and 32 and be selected from the VL district of SEQ ID NO:38 and 40.In some embodiments, CD3 binding domain comprise be selected from SEQ ID NO:28,30 and 32 VH district and be selected from SEQ ID NO:34,36, the VL district of 38 and 40.In some embodiments, VH district comprises SEQ ID NO:28 and VL district comprises SEQ ID NO:34; VH district comprises SEQ ID NO:28 and VL district comprises SEQ ID NO:38; VH district comprises SEQID NO:26 and VL district comprises SEQ ID NO:38; Or VH district comprises SEQ ID NO:26 and VL district comprises SEQ ID NO:34.In some embodiments, CD3 Binding peptide includes and comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3, and wherein heavy chain CDR3 comprises the CD3 binding domain of SEQ ID NO:51.In some embodiments, CD3 Binding peptide include comprise SEQ ID NO:49 heavy chain CDR1, comprise the CDR2 of SEQ ID NO:50 and comprise the CDR3 of SEQ ID NO:51, and comprise SEQ ID NO:52 light chain CDR1, comprise the CDR2 of SEQ ID NO:53 and comprise the CDR3 of SEQ ID NO:54.

In some embodiments, CD3 Binding peptide comprise be selected from SEQ ID NO:6,8,10,12,14,16, the aminoacid sequence of 18 and 20.

The present invention includes the multi-specific binding protein of the single chain polypeptide comprising dimerization, each single chain polypeptide comprises the first binding domain, N-terminal connexon, constant region for immunoglobulin, C-terminal connexon and CD3 binding domain of the present invention from amino to C-terminal.Equally, the present invention includes the multi-specific binding protein of the single chain polypeptide comprising dimerization, each single chain polypeptide comprises CD3 binding domain of the present invention, N-terminal connexon, constant region for immunoglobulin, C-terminal connexon and the first binding domain from amino to C-terminal.In some embodiments, N-terminal connexon can comprise immunoglobulin hinge region or may in fact consisting of.

In another aspect of the present invention, multi-specific binding protein comprises single chain polypeptide, and described single chain polypeptide comprises the first binding domain, N-terminal connexon, constant region for immunoglobulin, C-terminal connexon and CD3 binding domain from amino to C-terminal.Equally, the present invention includes multi-specific binding protein, it comprises CD3 binding domain, N-terminal connexon, constant region for immunoglobulin, C-terminal connexon and the first binding domain from amino to C-terminal.

The present invention includes multi-specific binding protein, described multi-specific binding protein comprises the first binding domain (such as, via the scFv that connexon is connected with another scFv) be connected with the second binding domain via connection subdomain.For example, the present invention includes multi-specific binding protein, it comprises and connects subdomain and CD3 binding domain (in V via peptide _h-connexon-V _lor V _l-connexon-V _horientation) the first binding domain of connecting is (in V _h-connexon-V _lor V _l-connexon-V _horientation).In some embodiments, dual specificity protein matter in scFv-connexon-scFv form can comprise the Weight variable territory and the territory that can lighten that stem from for the antibody of T cell antigen (such as CD3), and includes but not limited to variable domain disclosed herein.The connexon separating scFv territory can comprise ((Gly ₄) Ser) _n, wherein n=1-5 (SEQ ID NO:74-78).In one embodiment, connexon is ((Gly ₄) Ser) ₃(SEQ ID NO:76).Connexon can also comprise an about 8-12 amino acid.In some embodiments, in this form, the present protein of (" bispecific single-chain antibody ") does not comprise Fc district, and does not therefore have the relevant effector function of Fc.

The present invention comprises in fact the polypeptide containing binding domain as described in this article of any type.This polypeptide comprising antibody, its fragment, scFv, Fab, two scFv single domain antibody, double-chain antibody, two variable domain associated proteins and contain antibody or antibody fragment.There is described herein other type polypeptide included in the present invention.

In some embodiments, V _hand V _lterritory is used as scFv, and to allow scFv and V _h/ V _lmode in conjunction with target is connected with other polypeptide or aminoacid sequence/merges.In some embodiments, V as described in this article _hand V _lterritory is used in antibody or its fragment.For example, by described V _hand V _lterritory is incorporated into V _hand V _lthe natural place of territory in antibody.In some embodiments, CD3 Binding peptide of the present invention is antibody, such as, have V as described in this article _lchain and V _hchain.

In one embodiment of the invention, polyspecific protein is scFv dimer or double-chain antibody, instead of complete antibody.Double-chain antibody and scFv dimer can only use variable domain build and do not have Fc district.Double-chain antibody is bivalent, bispecific antibodies, wherein V _hand V _lterritory is expressed on Single polypeptide chain, but the peptide connexon used is too short and cannot allow two structural domain pairings on same chain, thus force the complementary territory of described structural domain and another chain to be matched and produce two antigen binding sites (see such as Holliger, P. etc. (1993) Proc.Natl.Acad.Sci.USA 90:6444-6448; Poljak, R.J. etc. (1994) Structure 2:1121-1123).Described antibody-binding fraction is well known in the art, and (Kontermann and Dubel compiles, Antibody Engineering (2001) Springer-Verlag.New York.790pp. (ISBN 3-540-41354-5).

In one embodiment of the invention, polyspecific protein is through the stable double-chain antibody of disulphide.For example, multi-specificity antibody can comprise two unique polypeptide, and their coexpressions have the covalently bound heterodimeric mixture of a binding site to each in 2 specificitys to produce.In this embodiment, each Fv is by being V _lA-V _hB(the first chain) and V _lB-V _hAv on a chain of (the second chain) configuration _lv on companion and second chain _hcompanion is associated and is formed.This can be stablized by any one in two replacement C-terminal heterodimeric territories: the VEPKSC (SEQ ID NO:254) on a chain matches with the FNRGEC (SEQ ID NO:255) on another chain, or the pairing of the coiled coil domain of oppositely charged.See such as Moore etc., 2011, Blood.117:4542-4551.In this embodiment, multi-specific binding protein can comprise and has and the first binding domain V _lthe CD3 binding domain V connected _hthe first chain, and the second chain comprises and the first binding domain V _hthe CD3 binding domain V connected _l, and two chains connect at C-terminal via disulfide linkage.The variable heavy chain and light chain that stem from known antibodies can be used, comprise variable heavy chain such as disclosed herein and light chain designs through the stable double-chain antibody of disulphide.

In another embodiment, multi-specific binding protein is can two variable domain associated proteins of specific binding first binding site (such as tumour antigen) and TCR mixture.In this embodiment, recombinant protein comprises polypeptide chain, wherein said polypeptide chain comprises VD1-(X1) n-VD2-C--(X2) n, wherein VD1 is the first variable domain, VD2 is the second variable domain, and C is constant domain, X1 be connexon (such as, the polypeptide linker of about 10 to 20 amino acid longs), X2 represents Fc district and n is 0 or 1.See such as U.S. Patent number 8,258,268.

In one embodiment of the invention, multi-specific binding protein comprises an one, two, three or more polypeptide chain.For example, the present invention includes polyspecific protein, it has and comprises V _h1-V _l2the first chain, comprise CH2-CH3-V _l1-V _h2the second chain and comprise the 3rd chain of CH2-CH3.In this embodiment, V _h1and V _l1the variable domain of the first binding domain can be corresponded to, and V _h2and V _l2antiCD3 McAb variable domain can be corresponded to.Or, V _h1and V _l1can AntiCD3 McAb variable domain be corresponded to, and V _h2and V _l2the variable domain of the first binding domain can be corresponded to.

In other embodiments, multi-specific binding protein is made up of the dimerization single chain polypeptide chain (heterodimer) comprising different single chain polypeptides.In this form (" polyspecific heterodimer "), each polypeptide chain comprises binding domain, N-terminal connexon, constant region, C-terminal connexon and another binding domain of optionally existing and also comprises heterodimeric territory.In some change programme, the second polypeptide chain for heterodimeric comprises other binding domain.Therefore, in certain embodiments of the invention, heterodimeric recombinant protein can containing two, three or four different binding domain.About some examples and methods involving, see PCT publication No. WO2011/090762.

As noted above, CD3 Binding peptide of the present disclosure comprises the binding domain of specific binding CD3.In some change programmes, CD3 binding domain can the CD3 with scFv (scFv) of competition binding aminoacid sequence as shown in SEQ ID NO:41.In certain embodiments, CD3 binding domain comprises the immunoglobulin heavy chain variable region (V that (i) comprises CDR HCDR1, HCDR2 and HCDR3 _h), and (ii) comprises the immunoglobulin light chain variable region (V of CDR LCDR1, LCDR2 and LCDR3 _l).Suitable CD3 binding domain comprises the V having and stem from murine monoclonal antibody Cris-7 _land V _hcD3 binding domain (Reinherz, E.L. etc. (volume), Leukocyte typing II., Springer Verlag, New York, (1986) in district.In the embodiment that some are such, LCDR3 has the aminoacid sequence described in SEQ ID NO:54 and/or HCDR3 has the aminoacid sequence described in SEQ ID NO:51; And LCDR1 and LCDR2 optionally has the aminoacid sequence as described in SEQ ID NO:52 and SEQ ID NO:53 respectively, and HCDR1 and HCDR2 optionally has the aminoacid sequence as described in SEQID NO:49 and SEQ ID NO:50 respectively.In some embodiments, for example, LCDR1, LCDR2 and LCDR3 have the aminoacid sequence respectively as shown in SEQ ID NO:52,53 and 54; And/or HCDR1, HCDR2 and HCDR3 have the aminoacid sequence respectively as shown in SEQ ID NO:49,50 and 51.

The exemplary anti-cd 3 antibodies that can obtain binding domain of the present disclosure comprises Cris-7 monoclonal antibody (Reinherz, E.L. etc. (volume), Leukocyte typing II., Springer Verlag, New York, (1986)

(V _l=QVVLTQSPAIMSAFPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDSSKLASG VPARFSGSGSGTSYSLTISSMETEDAATYYCQQWSRNPPTFGGGTKLQITR (SEQ ID NO:46) and

V _h=QVQLQQSGAELARPGASVKMSCKASGYTFTRSTMHWVKQRPGQGLEWIGYINPSSA YTNYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCASPQVHYDYNGFPYWG QGTLVTVSA (SEQID NO:45)); HuM291 (Chau etc. (2001) Transplantation 71:941-950 (V _l=DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKVEIK (SEQ ID NO:72) and

V _h=QVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQAPGQGLEWMGYINPRSG YTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARSAYYDYDGFAYWGQ GTLVTVSS (SEQID O:73); BC3 monoclonal antibody (Anasetti etc. (1990) J.Exp.Med.172:1691); OKT3 monoclonal antibody (Ortho muliicenier Transplant Study Group (1985) N.Engl.J.Med.313:337) and derivative thereof, such as OKT3ala-ala (also referred to as OKT3AA-FL or OKT3FL), namely has the humanization Fc variant (Herold etc. (2003) J.Clin.Invest.11:409) that L-Ala replaces on position 234 and 235; Tie up western pearl monoclonal antibody (Carpenter etc. (2002) Blood 99:2712); G19-4 monoclonal antibody (Ledbetter etc., 1986, J.Immunol.136:3945) and 145-2C11 monoclonal antibody (Hirsch etc. (1988) J.Immunol.140:3766).Exemplary T cell receptor is BMA031 monoclonal antibody (Borst etc. (1990) Human Immunology 29:175-188).

In some embodiments, binding domain comprises relevant target-specific V _hand V _lthe strand F in district _vfragment (scFv).In certain embodiments, V _hand V _ldistrict is people.

In one embodiment of the invention, the experience of CD3 Binding peptide and/or theoretical pI than DRA209SMIP little by least 0.25,0.5,0.75,1,1.25,1.5,2,2.5 an or more unit.In some embodiments, the experience of CD3 Binding peptide of the present invention and/or theoretical pI are than little about 0.25 to about 2.5,0.5 to about 2.5, about 0.5 to about 2.0, about 0.5 to about 1.5, about 0.5 to about 1.25, about 0.5 to about 1.0, about 0.5 to about 0.75, about 0.75 to about 1.0, about 0.75 to about 1.25, about 0.75 to about 1.5, about 0.75 to about 2.0, about 1.0 to about 1.5, about 1.0 to about 2.0 or about 1.0 to about 2.5 units of DRA209SMIP.In some embodiments, the experience of CD3 polypeptide of the present invention or theoretical pI are less than about 8.9,8.8,8.7,8.6,8.5,8.4,8.3,8.2,8.1,8,7.5,7.25,7.0 or 6.75.In some embodiments, the experience of CD3 polypeptide of the present invention or theoretical pI be about 8.9,8.8,8.7,8.6,8.5,8.4,8.3,8.2,8.1,8,7.5,7.25,7.27.0,6.8 or 6.75.In some embodiments, the experience of CD3 polypeptide of the present invention or theoretical pI are about 7.9 to about 8.7,7.9 to about 8.6,8.0 to about 8.6,8.0 to about 8.5,8.0 to about 8.4,8.0 to about 8.3,8.0 to about 8.2,8.0 to about 8.1, about 8.1 to about 8.6, about 8.2 to about 8.6, about 8.3 to about 8.6, about 8.4 to about 8.6, about 8.5 to about 8.6, about 8.5 to about 8.3, about 8.4 to about 8.2 or about 8.3 to about 8.1.

In certain embodiments, CD3 binding domain comprises or pI (experience and/or theory) reduces the scFv at least about 0.25,0.5,1,1.25,1.5,2,2.5 or more units compared with the scFv of SEQ ID NO:41.In some embodiments, CD3 binding domain comprise or pI (experience and/or theory) than the scFv of reduction about 0.25 to about 2.5,0.5 to about 2.5, about 0.5 to about 2.0, about 0.5 to about 1.5, about 0.5 to about 1.25, about 0.5 to about 1.0, about 0.5 to about 0.75, about 0.75 to about 1.0, about 0.75 to about 1.25, about 0.75 to about 1.5, about 0.75 to about 2.0, about 1.0 to about 1.5, about 1.0 to about 2.0 or about 1.0 to about 2.5 units of SEQ ID NO:41.Such scFv comprise SEQ ID NO:6,8,10,12,14,16, scFv contained in 18 and 20, or have at least about 90% with the aminoacid sequence of contained scFv in SEQ ID NO:6,8,10,12,14,16,18 and 20, scFv at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or 100% identity.

In some embodiments, CD3 binding domain of the present invention and SEQ ID NO:41 have about 80% to about 99%, about 82% to about 99%, about 84% to about 99%, about 86% to about 99%, about 88% to about 99%, about 90% to about 99%, about 92% to about 99%, about 94% to about 99%, about 96% to about 99%, about 97% to about 99%, about 98% to about 99%, about 80% to about 85%, about 85% to about 90%, about 85% to about 95%, about 90% to about 97%, about 90% to about 95%, about 90% to about 93%, about 90% to about 91%, about 95% to about 96%, about 96% to about 97%, about 97% to about 98%, the identity of about 96% to about 97% or about 96% to about 98%.

In some embodiments, CD3 binding domain of the present invention is the variant of SEQ ID NO:41, has wherein made the one or more amino acid mutations in aminoacid sequence to reduce the pI of binding domain.Sudden change comprises with another amino acid of aminoacid replacement to reduce the pI of binding domain, insertion amino acid to reduce the pI of binding molecule and/or delete amino acids to reduce the pI of binding domain.In one embodiment, sudden change replaces.In some embodiments, suddenly change in the framework region of variable chains.In some embodiments, 1,2,3,4,5,6,7,8,9,10 or more amino acid mutations are had in variant.

In one embodiment, CD3 binding domain comprise or comprise be selected from SEQ ID NO:28,30,32, the heavy chain of 38 and 40 or the scFv of light chain.In another embodiment, CD3 binding domain comprise be selected from SEQ ID NO:22,24,26,28, the VH district of 30 and 32 and be selected from the VL district of SEQ ID NO:38 and 40.In another embodiment, CD3 binding domain comprise be selected from SEQ ID NO:28,30 and 32 VH district and be selected from SEQ ID NO:34,36, the VL district of 38 and 40.In another embodiment, CD3 binding domain comprises SEQ ID NO:28 and 34.In another embodiment, CD3 binding domain comprises SEQ IDNO:28 and 38.In another embodiment, CD3 binding domain comprises SEQ ID NO:26 and 38.In another embodiment, CD3 binding domain comprises SEQ ID NO:26 and 34.In some embodiments, CD3 binding domain comprise the VL district that is selected from SEQ ID NO:38 and 40 and be selected from SEQ ID NO:22,24,26,28, the VH district of 30 and 32.In other embodiments, compared with the CDR of the monoclonal antibody or its fragment or derivative (such as CD3) that come from specific binding associated target, each CDR comprise be no more than one, two or three replace, insert or disappearance.

In some embodiments, CD3 binding domain in conjunction with the tcr complex in T cell CD3 ε subunit or with its interaction.In some embodiments, CD3 binding domain and Cris-7 or HuM291 monoclonal antibody competition binding CD3 ε.

In some embodiments, CD3 Binding peptide inducing T cell receptor complex internalization.

In some change programmes, binding domain comprises the immunoglobulin (Ig) V engaged by peptide connexon _ldistrict and V _hthe scFv (scFv) in district.For engaging V _ldistrict and V _hthe use of the peptide connexon in district is well-known in the art, and there is many announcements in this specific area.A kind of widely used peptide connexon is Gly-Gly-Gly-Gly-Ser (SEQ IDNO:74) aminoacid sequence (Gly repeated by 3 ₄ser) ₃15 aggressiveness that (EQ ID NO:76) forms.In some embodiments, peptide connexon comprise in SEQ ID NO:74-78 any one or consisting of.Employ other connexon, and use display technique of bacteriophage and the infectious phage technology of selectivity make the variation of connexon sequence and select suitable connexon sequence (Tang etc., J.Biol.Chem.271,15682-15686,1996; Hennecke etc., ProteinEng.11,405-410,1998).In certain embodiments, V _hdistrict and V _ldistrict is by having contained (Gly ₄ser) _naminoacid sequence peptide connexon be connected, wherein n=1-5 (being SEQ ID NO:74-78 respectively).Can occur to optimize simple connexon (such as (Gly by random mutation ₄ser) _n) obtain other suitable connexon.

In certain embodiments, binding domain comprises Humanized immunoglobulin V _land/or V _hdistrict.For making immunoglobulin (Ig) V _ldistrict and V _hthe humanized technology in district is well known in the art, and is discussed in such as U.S. Patent Application Publication No. 2006/0153837.

Expect that " humanization " produces less immunogenic, retains the antibody of the antigen-binding matter of initial molecule completely.In order to retain all antigen-binding matter of original antibodies, " humanization " pattern of its antigen binding site structure will be produced again.This can pass through in reservation or only inhuman CDR is transplanted to (Jones etc., Nature 321:522 (1986) in the variable framework territory of people and constant region when not retaining important Framework residues; Verhoeyen etc., Science 239:1539 (1988)) or pass through the whole inhuman variable domain of restructuring (to retain ligand binding property) but by the residue proper manners surface of replacing exposure carefully, it " is sheltered " (to reduce antigenicity) (Padlan, Molec.Immunol.28:489 (1991)) to realize.

In fact, transplanted by CDR and carry out humanization and relate to and only the CDR of non-human antibody being recombinated on people variable region framework and human constant region.In theory, this will significantly reduce or eliminate immunogenicity (unless there is allotype or idiotype difference).But, report some Framework residues (Reichmann etc., Nature, the 332:323 (1988) that also may need to retain original antibodies; Queen etc., Proc.Natl.Acad.Sci.USA, 86:10,029 (1989)).

The Framework residues retained is needed to be suitable for being differentiated by computer simulation.Or, important Framework residues (Padlan can be differentiated potentially by more known antigen binding site structure, Molec.Immunol., 31 (3): 169-217 (1994), be incorporated herein by reference).

The residue affecting antigen combination is potentially divided into some groups.First group comprises the continuous residue with antigen site surface, and therefore they directly can contact with antigen.These residues comprise n terminal residue and the residue adjacent with CDR.Second group comprises and can change the structure of CDR or the residue of arranged opposite by the CDR in contact antibody or another peptide chain.3rd group comprises and has the amino acid that possibility affects the hiding side chain of the structural integrity of variable domain.(Padlan, 1994, the same) that residue in these groups normally finds on identical position, but depend on numbering system, their positions when differentiating may different (see Kabat etc., " Sequencesof proteins of immunological interest, the 5th edition; issue number 91-3242; U.S.Dept.Health & Human Services, NIH, Bethesda; Md., 1991).

Although examples more described herein relate to the humanization of scFv, SMIP, Scorpion and Interceptor molecule instead of antibody, be applicable to according to polypeptide of the present invention about the knowledge of humanized antibody in this area.

In certain embodiments, hinge is wild type human immunoglobulin hinge region.In some other embodiment, the part that one or more amino-acid residue designs as fusion protein construct can be added on the amino of wild-type immunoglobulin hinge region or C-terminal.For example, it can be " RT ", " RSS ", " TG " or " T " that aminoterminal other of hinge engages amino-acid residue, or can be " SG " at hinge C-terminal, maybe hinge disappearance can combine with adding, such as there is in C-terminal interpolation the Δ Ρ of " SG ".

In certain embodiments, hinge is the immunoglobulin (Ig) hinge changed, and the one or more cysteine residues wherein in wild-type immunoglobulin hinge area replace through other amino-acid residue one or more (such as Serine or L-Ala).

The immunoglobulin (Ig) hinge of exemplary change includes but not limited to exist find in wild type human IgG1 hinge one, two or three cysteine residues are by one, the immunoglobulin (Ig) human IgG1 hinge area that replaces of two or three different amino-acid residues (such as Serine or L-Ala).Proline(Pro) may be there is in addition and replaced by another amino acid (such as Serine or L-Ala) in the immunoglobulin (Ig) hinge changed.For example, can there is the proline(Pro) being connected to three halfcystines of wild type human IgG1 hinge area being positioned at C-terminal in addition and replaced by another amino-acid residue (such as Serine, L-Ala) in human IgG1's hinge of above-mentioned change.In one embodiment, the proline(Pro) of core hinge area is unsubstituted.

In certain embodiments, hinge polypeptide comprises or has the sequence of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with wild-type immunoglobulin hinge areas such as such as wild type human IgG1 hinge, wild type human IgG2 hinge or wild type human IgG4 hinges.

In other embodiments, the hinge existed in CD3 Binding peptide can be not based on or stem from the hinge (that is, the immunoglobulin (Ig) hinge of non-wild-type immunoglobulin hinge or change) of immunoglobulin (Ig) hinge.What the example of such hinge comprised the district of stem of Yu Jian district or the II type C-lectin stemming from transmembrane protein has about 5 to about 150 amino acid whose peptides, such as, have about 8 to 25 amino acid whose peptides and have about 7 to 18 amino acid whose peptides.

In certain embodiments, Yu Jian district or stem's district's hinge have 7 to 18 amino acid, and can form alpha-helix coiled-coiled structure.In certain embodiments, Yu Jian district or stem's district's hinge contain 0,1,2,3 or 4 halfcystine.The peptide fragment in district or hinge Shi Yujian district of district of stem or district of stem between example domain, such as comes from 10 to 150 amino acid whose fragments in the district of stem of CD69, CD72, CD94, NKG2A and NKG2D.

In certain embodiments, hinge sequence has about 5 to 150 amino acid, 5 to 10 amino acid, 10 to 20 amino acid, 20 to 30 amino acid, 30 to 40 amino acid, 40 to 50 amino acid, 50 to 60 amino acid, 5 to 60 amino acid, 5 to 40 amino acid, 8 to 20 amino acid or 10 to 15 amino acid.Hinge can be mainly flexible, but also can provide more rigidity characteristic, or mainly can contain α-helixstructure and few β-sheet structure.It is active that the length of hinge or sequence may affect one or more of the direct or indirect Fc district part that directly or indirectly connects of (via another district or territory, such as heterodimeric territory) binding affinity of binding domain of connecting and hinge of hinge.

In certain embodiments, hinge sequence is stable and can resists proteolytic cleavage in blood plasma and serum.The first lysine mutation in IgG1 upper hinge district can be made to be minimized by proteolytic cleavage, and for example, Methionin can replace or disappearance through methionine(Met), Threonine, L-Ala or glycine.

In some embodiments of the present invention, CD3 Binding peptide can form heterodimer with the second polypeptide chain and comprise following hinge area: (a) N-terminal is close to constant region for immunoglobulin (such as, N-terminal is connected to CH2 territory, wherein constant region for immunoglobulin comprises CH2 and CH3 territory, or N-terminal is connected to CH3 territory, wherein immunoglobulin domain comprises CH3 and CH4 territory); B () to be inserted between binding domain (such as scFv) with immunoglobulin (Ig) heterodimeric territory and to be connected binding domain and immunoglobulin (Ig) heterodimeric territory; C () to be inserted between immunoglobulin (Ig) heterodimeric territory and constant region for immunoglobulin (such as, wherein constant region for immunoglobulin comprises CH2 and CH3 territory or CH3 and CH4 territory) and to connect immunoglobulin (Ig) heterodimeric territory and constant region for immunoglobulin; D () to be inserted between constant region for immunoglobulin with binding domain and to be connected constant region for immunoglobulin and binding domain; E () is at the N-terminal of polypeptide chain; Or (f) is at the C-terminal of polypeptide chain.The polypeptide chain comprising hinge area as described in this article can associate to form provided homologous dimerization or heterodimeric albumen herein from different polypeptide chains, and the dimer formed is by containing the binding domain remaining its target-specific and/or its specific target binding affinity.

In certain embodiments, can form with another polypeptide chain the hinge existed in the polypeptide of heterodimer can be immunoglobulin (Ig) hinge, such as wild-type immunoglobulin hinge area or its immunoglobulin hinge region changed.In certain embodiments, the hinge of a polypeptide chain of heterodimeric albumen is same with the corresponding hinge of another polypeptide chain of described heterodimer.In some other embodiment, the hinge of a chain is different from the hinge of another chain (such as, in its length and/or sequence).Different hinge in different chains allows to carry out different control to the binding affinity of the binding domain that hinge connects, and makes heterodimer can skip over the target of another binding domain in conjunction with the target of a binding domain.For example, in certain embodiments, heterodimeric albumen has CD3 binding domain or TCR binding domain in a chain, and in another chain, have the second binding domain (such as tumour antigen binding domain).In two chains, have two different hinges can allow heterodimer first in conjunction with tumour antigen, then in conjunction with CD3 or other TCR component.Thus, heterodimer can by CD3 ⁺t cell raises the cell (such as, expressing the tumour cell of PSMA) of expressing tumor antigen, and this can destroy or damage again the cell of expressing tumor antigen.

Be applicable to exemplary hinge district used according to the invention to be shown in in following table 1 and 2.

Table 1: exemplary hinge district

Table 2: exemplary hinge district (be obtained from H7 hinge, the district of stem of II type C-lectin, or the Yu Jian district of I type transmembrane protein)

In certain embodiments, CD3 Binding peptide of the present invention or protein can comprise in " immunoglobulin (Ig) dimerization territory " or " immunoglobulin (Ig) heterodimeric territory ".

" immunoglobulin (Ig) dimerization territory " or " immunoglobulin (Ig) heterodimeric territory " refers to the immunoglobulin domain of the polypeptide chain interacting with the second immunoglobulin domain of the second polypeptide chain or associate as used herein, wherein the interaction in the first and second immunoglobulin (Ig) heterodimeric territories substantially contributes to or can effectively promote the heterodimeric of the first and second polypeptide chains (namely, between two different polypeptide chains, form dimer, this is also referred to as " heterodimer " or " heterodimeric albumen ").If when there is not the immunoglobulin (Ig) heterodimeric territory in the immunoglobulin (Ig) heterodimeric territory of the first polypeptide chain and/or the second polypeptide chain, there is statistically evident minimizing in the dimerization of the first and second polypeptide chains, then the interaction between immunoglobulin (Ig) heterodimeric territory " contributes to or can effectively promote " heterodimeric of the first and second polypeptide chains substantially.In certain embodiments, when the first and second polypeptide chain coexpressions, at least 60%, the first and second polypeptide chains at least about 60% to about 70%, at least about 70% to about 80%, at least 80% to about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with form heterodimer each other.Representative immunoglobulin (Ig) heterodimeric territory comprises immunoglobulin (Ig) CH1 territory, immunoglobulin (Ig) CL territory (such as, C κ or C λ isotype) or derivatives thereof, comprise immunoglobulin (Ig) CH1 and the CL territory of wild-type immunoglobulin CH1 and CL territory and change (or sudden change), such as provided herein.

Can use dimerization/heterodimeric territory when needs form heterodimer by two non-same polypeptide chains, wherein one or two polypeptide chain comprises binding domain.In certain embodiments, a polypeptide chain member of some heterodimer described herein is not containing binding domain.As noted above, heterodimeric albumen of the present disclosure comprises immunoglobulin (Ig) heterodimeric territory in each polypeptide chain.Immunoglobulin (Ig) heterodimeric territory in the polypeptide chain of heterodimer is different from each other, and thus can carry out to otherness modifying to promote that the heterodimeric of two chains and the homologous dimerization by any one chain minimize.As shown in PCT publication No. WO2011/090762, immunoglobulin (Ig) heterodimeric territory provided herein allows, between different polypeptide, effective heterodimeric occurs and promotes the purifying of gained heterodimeric albumen.

As herein provide, can be used for promoting that immunoglobulin (Ig) heterodimeric territory heterodimeric occurring according to two different single chain polypeptides of the present disclosure (such as, short long) comprises immunoglobulin (Ig) CH1 and CL territory, such as people CH1 and CL territory.In certain embodiments, immunoglobulin (Ig) heterodimeric territory is wild-type CH1 territory, such as wild-type IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE or IgM CH1 territory.In other embodiments, immunoglobulin (Ig) heterodimeric territory be announce No.WO2011/090762 as PCT respectively the wild type human IgG1 described in SEQ ID NO:114,186-192 and 194 (as described in sequence be incorporated herein by reference), IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE or IgM CH1 territory.In certain embodiments, immunoglobulin (Ig) heterodimeric territory is the wild type human IgG1CH1 territory as described in the SEQ ID NO:114 (as described in sequence be incorporated herein by reference) of WO2011/090762, and described sequence is identical with the SEQ IDNO:80 of the application.

In other embodiments, immunoglobulin (Ig) heterodimeric territory is the immunoglobulin (Ig) CH1 territory changed, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE or IgM CH1 territory such as changed.In certain embodiments, immunoglobulin (Ig) heterodimeric territory is human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE or IgM CH1 territory of change.In other embodiments, in the immunoglobulin (Ig) CH1 territory changed, the cysteine residues participating in the wild-type CH1 territory (such as people CH1) forming disulfide linkage together with wild-type immunoglobulin CL territory (such as people CL) lacks or is substituted, and makes not form disulfide linkage between the CH1 territory of change and wild-type CL territory.

In certain embodiments, immunoglobulin (Ig) heterodimeric territory is wild-type CL territory, such as wild-type C κ territory or wild-type C λ territory.In certain embodiments, immunoglobulin (Ig) heterodimeric territory is wild type human C κ respectively as described in the SEQ ID NO:112 and 113 (as described in sequence be incorporated herein by reference) of WO2011/090762 or people C λ territory, and described sequence is identical with 82 with the SEQ ID NO:81 of the application respectively.In other embodiments, immunoglobulin (Ig) heterodimeric territory is the immunoglobulin (Ig) CL territory changed, the C κ such as changed or C λ territory, such as, and the people C κ of change or people C λ territory.

In certain embodiments, in the immunoglobulin (Ig) CL territory changed, the cysteine residues participating in the wild-type CL territory (such as people CL) forming disulfide linkage together with wild-type immunoglobulin CH1 territory (such as people CH1) lacks or is substituted.The CL territory of described change can also comprise aminoacid deletion at its N-terminal.Exemplary C κ territory is set forth in the SEQ ID NO:141 of WO2011/090762, and (described sequence is incorporated herein by reference, it is the SEQ ID NO:83 of the application) in, wherein first arginine in wild type human C κ territory all lacks with last halfcystine.In certain embodiments, in the Ck territory changed, only last halfcystine disappearance in wild type human Ck territory, arginine can be had by C-terminal this is because first arginine lacks from wild type human Ck territory and connect the N-terminal in the Ck territory changed and another structural domain (such as, immunoglobulin (Ig) subprovince, such as comprises the subprovince in immunoglobulin (Ig) CH2 and CH3 territory) connexon provide.Exemplary C λ territory is set forth in the SEQ ID NO:140 of WO2011/090762, and (described sequence is incorporated herein by reference, it is the SEQ ID NO:84 of the application) in, wherein first arginine depletion in wild type human C λ territory and the halfcystine participating in being formed disulfide linkage together with the halfcystine in CH1 territory are replaced by Serine.

In other embodiments, immunoglobulin (Ig) heterodimeric territory be may participate in being formed interchain hydrogen bond network on C κ-C κ interface compared with wild-type C κ territory position on the C κ territory of change containing one or more aminoacid replacement.For example, in certain embodiments, immunoglobulin (Ig) heterodimeric territory on position N29, N30, Q52, V55, T56, S68 or T70, has one or more amino acid by the people C κ territory of the change of different aminoacid replacement.Amino acid whose numbering is based on its position in the people C κ sequence of the change as described in the SEQ ID NO:141 (as described in sequence be incorporated herein by reference, it is the SEQ ID NO:83 of the application) of WO2011/090762.In certain embodiments, immunoglobulin (Ig) heterodimeric territory is the people C κ territory of the change having one, two, three or four aminoacid replacement on position N29, N30, V55 or T70.Amino acid as the substituent on above-mentioned position can be L-Ala or the amino-acid residue with base side chain portion, such as arginine, tryptophane, tyrosine, L-glutamic acid, glutamine or Methionin.Other amino-acid residue that can be used for the amino-acid residue replaced in wild type human Ck sequence on above-mentioned position (such as N30) comprises aspartic acid, methionine(Met), Serine and phenylalanine.The people C κ territory of exemplary change is set forth in (described sequence is incorporated herein by reference) in the SEQ ID NO:142-178 of WO2011/090762.The people C κ territory changed promotes to carry out heterodimeric with CH1 territory, but the C κ territory that the homologous dimerization with another C κ territory is minimized.The representative people C κ territory changed is set forth in the SEQ ID NO:160 (N29W V55A T70A) of WO2011/090762, 161 (N29Y V55A T70A), 202 (T70E N29A N30A V55A), 167 (N30RV55A T70A), 168 (N30K V55A T70A), 170 (N30E V55A T70A), 172 (V55R N29A N30A), 175 (N29W N30Y V55A T70E), 176 (N29Y N30YV55A T70E), 177 (N30E V55A T70E), 178 (N30Y V55A T70E), 838 (N30D V55A T70E), 839 (N30M V55A T70E), in 840 (N30S V55A T70E) and 841 (N30F V55A T70E) (described sequence is incorporated herein by reference).

In certain embodiments, except the sudden change in Ck territory described herein or as its sub, the immunoglobulin (Ig) heterodimeric territory (such as immunoglobulin (Ig) CH1 and CL territory) of polypeptide heterodimer has sudden change, make to form salt bridge (that is, ionic interaction) between the amino-acid residue of gained immunoglobulin (Ig) heterodimeric territory on mutational site.For example, the immunoglobulin (Ig) heterodimeric territory of polypeptide heterodimer can be the combination in the CH1 territory of sudden change and the Ck territory of sudden change.In the CH1 territory of sudden change, α-amino-isovaleric acid (V68) on the position 68 in wild type human CH1 territory is had the amino-acid residue of negative charge (such as aspartic acid or L-glutamic acid) and is replaced, and the leucine (L29) on the position 29 in the people Ck territory of the sudden change of first arginine and last halfcystine disappearance is had the amino-acid residue of positive charge (such as Methionin, arginine or Histidine) replacement.Have the amino-acid residue of negative charge and the gained charge-charge suddenlyd change between the amino-acid residue in Ck territory with positive charge in gained sudden change CH1 territory to interact and form salt bridge, described salt bridge can make the heterodimeric interface stability between CH1 and the Ck territory of sudden change.Or the V68 of wild-type CH1 can be replaced by the amino-acid residue with positive charge, and the L29 in the mutant human Ck territory of first arginine and last halfcystine disappearance can be replaced by the amino-acid residue with negative charge.V68 is set forth in (described sequence is incorporated herein by reference) in the SEQ IDNO:844 and 845 of WO2011/090762 by the exemplary mutations CH1 sequence with the aminoacid replacement of positive charge or negative charge.L29 is set forth in (described sequence is incorporated herein by reference) in the SEQ ID NO:842 and 843 of WO2011/090762 by the exemplary mutations Ck sequence with the aminoacid replacement of positive charge or negative charge.

Except the sudden change in the L29 in V68 and the Ck territory in CH1 territory or as its alternative scheme, the position except the V68 in people CH1 territory and the L29 in people Ck territory also can be had the aminoacid replacement of opposite charges to produce ionic interaction between amino acid.Described position can be differentiated by any suitable method, comprises random mutation and occurs; Analyze the crystalline structure of CH1-Ck pairing, this is for differentiating amino-acid residue on CH1-Ck interface and use in the amino-acid residue of one group of standard (such as, participate in the proneness of ionic interaction, with proximity of potential companion's residue etc.) on CH1-Ck interface to differentiate suitable position further.

In certain embodiments, polypeptide heterodimer of the present disclosure is only containing a pair immunoglobulin (Ig) heterodimeric territory.For example, first chain of polypeptide heterodimer can comprise CH1 territory as immunoglobulin (Ig) heterodimeric territory, and second chain can comprise CL territory (such as C κ or C λ) as immunoglobulin (Ig) heterodimeric territory.Or first chain can comprise CL territory (such as C κ or C λ) as immunoglobulin (Ig) heterodimeric territory, and second chain can comprise CH1 territory as immunoglobulin (Ig) heterodimeric territory.As described herein, the immunoglobulin (Ig) heterodimeric territory of first and second chain can associate to form heterodimeric albumen of the present disclosure.

In some other embodiment, heterodimeric albumen of the present disclosure can have two pairs of immunoglobulin (Ig) heterodimeric territories.For example, first chain of heterodimer can comprise two CH1 territories, and second chain can have two CL territories, associates in two CH1 territories of these two CL territories and first chain.Or first chain can comprise two CL territories, and second chain can have two CH1 territories, associates in two CL territories of these two CH1 territories and first chain.In certain embodiments, first polypeptide chain comprises CH1 territory and CL territory, and second polypeptide chain comprises CL territory and CH1 territory.Associate with the CH1 territory of first polypeptide chain and CL territory respectively in this CL territory and CH1 territory.

A heterodimeric pairing is only comprised (such as at heterodimeric albumen, have an immunoglobulin (Ig) heterodimeric territory in each chain) embodiment in, the immunoglobulin (Ig) heterodimeric territory of each chain can be positioned at the N-terminal of the immunoglobulin (Ig) constant sub-region of this chain.Or the immunoglobulin (Ig) heterodimeric territory in each chain can be positioned at the C-terminal of the immunoglobulin (Ig) constant sub-region of this chain.

Two heterodimeric pairings are comprised (such as at heterodimeric albumen, have two immunoglobulin (Ig) heterodimeric territories in each chain) embodiment in, two immunoglobulin (Ig) heterodimeric territories in each chain can be positioned at the N-terminal of the immunoglobulin (Ig) constant sub-region of this chain.Or two immunoglobulin (Ig) heterodimeric territories in each chain all can be positioned at the C-terminal of the immunoglobulin (Ig) constant sub-region of this chain.In other embodiments, an immunoglobulin (Ig) heterodimeric territory in each chain can be positioned at the N-terminal of the immunoglobulin (Ig) constant sub-region of this chain, and another immunoglobulin (Ig) heterodimeric territory can be positioned at the C-terminal of the immunoglobulin (Ig) constant sub-region of this chain.In other words, in those embodiments, immunoglobulin (Ig) constant sub-region be inserted in each chain two immunoglobulin (Ig) heterodimeric territories between.

As indicated, in certain embodiments, CD3 Binding peptide of the present disclosure (such as little module formula immune drug albumen) (SMIP), homodimer dual specific therapeutical agent are (such as, Scorpion), heterodimer monospecific and polyspecific therapeutical agent (such as, Interceptor) comprise constant region for immunoglobulin in each polypeptide chain.Term " constant region " is used interchangeably in this article with constant sub-region and constant region for immunoglobulin.Comprise constant region for immunoglobulin and can slow down the removing from circulation after being administered to experimenter by the homologous dimerization of two CD3 Binding peptide chain formation and heterodimeric albumen.

Changed by sudden change or other, constant region for immunoglobulin also makes it possible to relatively easily regulate dimeric polypeptide effector function (such as, ADCC, ADCP, CDC, complement fixation and Fc receptors bind), as known in the art with described herein, described dimeric polypeptide effector function can depend on treated disease and increase or reduce.

In certain embodiments, one of the polypeptide chain of homologous peptide dimer of the present disclosure and heterodimer or both constant region for immunoglobulin should be able to mediate one or more in these effector functions.

In other embodiments, compared with corresponding wild-type immunoglobulin constant region, in one of the polypeptide chain or both constant region for immunoglobulin of homologous peptide dimer of the present disclosure and heterodimer, one or more in these effector functions are lowered or lack.For example, monospecific CD3 Binding peptide and the constant region for immunoglobulin of CD3 Binding peptide comprising the second binding domain (such as, be designed for cause be redirected T cell toxicity (RTCC)) have the effector function of reduction relative to the wild-type immunoglobulin constant region of correspondence or are useful especially without effector function.In one embodiment, constant region is modified so that not complement-fixing.Constant region can also be modified so that not in conjunction with one or more Fc γ acceptors, such as CD16, CD32 and CD64.

The immunoglobulin (Ig) constant sub-region existed in CD3 Binding peptide of the present disclosure can be made up of the following or stem from part or all of the following: CH2 territory, CH3 territory, CH4 territory or its any combination.For example, immunoglobulin (Ig) constant sub-region can comprise the part in CH2 territory, CH3 territory, CH2 territory and CH3 territory, CH3 territory and CH4 territory, two CH3 territories, CH4 territory, two CH4 territories and CH2 territory and CH3 territory.In some embodiments, constant region comprises CH2 and the CH3 territory of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD or its any combination; The immunoglobulin (Ig) CH3 territory of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, IgM or its any combination; IgE, IgM or its combination immunoglobulin (Ig) CH3 and CH4 territory.In some embodiments, constant region is made up of CH2 territory and CH3 territory in fact.

The CH2 territory that can form the constant region for immunoglobulin of CD3 Binding peptide of the present disclosure can be come from some immunoglobulin class or subclass (such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2 or IgD) and come from the wild-type immunoglobulin CH2 territory of various species (comprising people, mouse, rat and other Mammals) or the immunoglobulin (Ig) CH2 territory of its change.In some embodiments, constant region modified with reduce or eliminate effector function, minimizing or not complement-fixing and/or reduce combine or not in conjunction with Fc γ acceptor.In some embodiments, Fc γ acceptor is selected from CD16, CD32 and CD64.

In certain embodiments, CH2 territory is wild type human immunoglobulin (Ig) CH2 territory, the wild-type CH2 territory of such as human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2 or IgD, respectively as PCT announces described in SEQ ID NO:115,199-201 and 195-197 (as described in sequence be incorporated herein by reference) of WO2011/090762.In certain embodiments, CH2 territory is wild type human IgG1CH2 territory as described in the SEQ ID NO:115 of WO2011/090762 (as described in sequence be incorporated herein by reference, it is the SEQ ID NO:85 of the application).In some embodiments, constant region comprise SEQ ID NO:55 aminoacid sequence or consisting of.

In certain embodiments, CH2 territory is the immunoglobulin (Ig) CH2 district (such as, the human IgG1 CH2 territory of change) of the change comprising aminoacid replacement (such as, l-asparagine becomes L-Ala) on the l-asparagine of position 297.Such aminoacid replacement reduces or eliminates the glycosylation on this site and cancels effective Fc combination of Fc γ R and C1q.Position 297 has the sequence in human IgG1 CH2 territory that Asn becomes the change that Ala replaces and is set forth in (described sequence is incorporated herein by reference, and it is the SEQ ID NO:86 of the application) in the SEQ ID NO:324 of WO2011/090762.

In certain embodiments, CH2 territory is the immunoglobulin (Ig) CH2 district (such as, the human IgG1 CH2 territory of change) of the change comprising at least one replacement or disappearance on position 234 to 238.For example, immunoglobulin (Ig) CH2 district other combination any of on position 234,235,236,237 or 238, position 234 and 235, position 234 and 236, position 234 and 237, position 234 and 238, position 234-236, position 234,235 and 237, position 234,236 and 238, position 234,235,237 and 238, position 236-238 or on the 234-238 of position two, three, four or five amino acid can comprise replacement.In addition or instead scheme, the CH2 district changed can on the 234-238 of position, on one of position 236 or position 237, such as comprise one or more (such as two, three, four or five) aminoacid deletion, and be substituted on another position.Said mutation can reduce or eliminate the active or Fc receptor binding capacity of the cytotoxicity (ADCC) of antibody dependent cellular mediation of polypeptide heterodimer in the CH2 territory comprising change.In certain embodiments, with the amino-acid residue on one or more in one or more alanine residue displacement position 234-238.In other embodiments, an only disappearance in the amino-acid residue on the 234-238 of position, and one or more in all the other amino acid on the 234-238 of position can replace through another amino acid (such as L-Ala or Serine).

In some other embodiment, CH2 territory is the immunoglobulin (Ig) CH2 district (such as, the human IgG1 CH2 territory of change) comprising the change of one or more aminoacid replacement on position 253,310,318,320,322 and 331.For example, immunoglobulin (Ig) CH2 district on position 253,310,318,320,322 or 331, position 318 and 320, position 318 and 322, position 318,320 and 322 or on position 253,310,318,320,322 and 331 two, three, four, five or six amino acid whose other combinations any can comprise replacement.Said mutation can reduce or eliminate the CDC (CDC) of polypeptide heterodimer in the CH2 territory comprising change.

In some other embodiment, except the aminoacid replacement on position 297, the CH2 district (such as, the human IgG1 CH2 territory of change) changed can also comprise one or more (such as, two, three, four or five) on the 234-238 of position, and other replaces.For example, immunoglobulin (Ig) CH2 district any combination of on position 234 and 297, position 234,235 and 297, position 234,236 and 297, position 234-236 and 297, position 234,235,237 and 297, position 234,236,238 and 297, position 234,235,237,238 and 297, position 236-238 and 297 or the position 234-238 except position 297 two, three, four or five amino acid can comprise replacement.In addition or instead scheme, the CH2 district of change on the 234-238 of position, such as can comprise one or more (such as two, three, four or five) aminoacid deletion on position 236 or position 237.Cytotoxicity (ADCC) activity of the antibody dependent cellular mediation of other polypeptide heterodimer that can reduce or eliminate the CH2 territory comprising change that suddenlys change or Fc receptor binding capacity.In certain embodiments, with the amino-acid residue on one or more in one or more alanine residue displacement position 234-238.In other embodiments, an only disappearance in the amino-acid residue on the 234-238 of position, and one or more in all the other amino acid on the 234-238 of position can replace through another amino acid (such as L-Ala or Serine).

In certain embodiments, except on the 234-238 of position one or more (such as, 2,3,4 or 5) beyond aminoacid replacement, the CH2 district of the sudden change in fusion rotein of the present disclosure (such as, the human IgG1 CH2 territory changed) can on the position of one or more participation complement fixation (such as, on position I253, H310, E318, K320, K322 or P331) containing one or more (such as, 2,3,4,5 or 6) other aminoacid replacement (such as replacing through L-Ala).The example in immunoglobulin (Ig) CH2 district of sudden change be included in position 234,235,237 (if existence), 318,320 and 322 has L-Ala and replaces human IgG1, IgG2, IgG4 and mouse IgG 2a CH2 district.The immunoglobulin (Ig) CH2 district of exemplary mutations is the mouse IGHG2c CH2 district on L234, L235, G237, E318, K320 and K322 with L-Ala replacement.

In other embodiments, except other disappearance on the aminoacid replacement on position 297 and position 234-238 or replacement, the CH2 district changed (such as, the human IgG1 CH2 territory changed) other replaces can also to comprise one or more (such as, two, three, four, five or six) on position 253,310,318,320,322 and 331.For example, immunoglobulin (Ig) CH2 district can comprise the replacement on (1) position 297, (2) the one or more replacement on the 234-238 of position or disappearance or its combination, with on position I253, H310, E318, K320, K322 and P331 one or more (such as, 2,3,4,5 or 6) aminoacid replacement, one on such as position E318, K320 and K322, two, three replacements.Amino acid on above-mentioned position can be replaced by L-Ala or Serine.

In certain embodiments, immunoglobulin (Ig) CH2 district polypeptide comprises: an aminoacid replacement on the aminoacid replacement on the l-asparagine of (i) position 297 and position 234,235,236 or 237; (ii) aminoacid replacement on two in the aminoacid replacement on the l-asparagine of position 297 and position 234-237; (iii) aminoacid replacement on three in the aminoacid replacement on the l-asparagine of position 297 and position 234-237; (iv) aminoacid replacement, the aminoacid replacement on position 234,235 and 237 and the aminoacid deletion on position 236 on the l-asparagine of position 297; Aminoacid replacement on aminoacid replacement on three in the 234-237 of (v) position and position 318,320 and 322; Or aminoacid replacement, the aminoacid deletion on position 236 and the aminoacid replacement on position 318,320 and 322 in (vi) position 234-237 three.

The immunoglobulin (Ig) CH2 district that the l-asparagine of position 297 has the exemplary change of aminoacid replacement comprises: at L234, L235, G237 and N297 has human IgG1 CH2 district (the SEQ IDNO:325 of WO2011/090762 that L-Ala replaces and have disappearance on G236, described sequence is incorporated herein by reference), at V234, G236 and N297 has human IgG2 CH2 district (the SEQ IDNO:326 of WO2011/090762 that L-Ala replaces, described sequence is incorporated herein by reference), at F234, L235, G237 and N297 has human IgG 4CH2 district (the SEQ ID NO:322 of WO2011/090762 that L-Ala replaces and have disappearance on G236, described sequence is incorporated herein by reference), F234 and N297 has human IgG 4CH2 district (the SEQ ID NO:343 of WO2011/090762 that L-Ala replaces, described sequence is incorporated herein by reference), L235 and N297 has human IgG 4CH2 district (the SEQ ID NO:344 of WO2011/090762 that L-Ala replaces, described sequence is incorporated herein by reference), G236 and N297 has human IgG 4CH2 district (the SEQ ID NO:345 of WO2011/090762 that L-Ala replaces, described sequence is incorporated herein by reference), with human IgG 4CH2 district (the SEQ ID NO:346 of WO2011/090762 on G237 and N297 with L-Ala replacement, described sequence is incorporated herein by reference).

In certain embodiments, except above-mentioned aminoacid replacement, other aminoacid replacement one or more may be contained on the one or more positions except above-mentioned position in the CH2 district (such as, the human IgG1 CH2 territory of change) of change.Described aminoacid replacement can be conservative or nonconserved amino acid replaces.For example, in certain embodiments, in the IgG2CH2 district changed, P233 can become E233 (see the SEQ IDNO:326 of such as WO2011/090762, described sequence is incorporated herein by reference).In addition or instead scheme, in certain embodiments, the CH2 district of change can contain one or more aminoacid insertion, disappearance or both.Insert, lack or replace and can be arranged in immunoglobulin (Ig) CH2 district Anywhere, such as at N or the C-terminal in wild-type immunoglobulin CH2 district, thus via chain connection CH2 district and another district (such as, binding domain or immunoglobulin (Ig) heterodimeric territory).

In certain embodiments, the CH2 district of the change in polypeptide of the present disclosure comprise or with wild-type immunoglobulin CH2 district, the CH2 district of such as wild type human IgG1, IgG2 or IgG4 or mouse IgG 2a (such as, IGHG2c) has the sequence of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity.

The immunoglobulin (Ig) CH2 district of the change in CD3 Binding peptide of the present disclosure can stem from the various Immunoglobulin Isotypes of various species (comprising people, mouse, rat and other Mammals), the CH2 district of such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2 and IgD.In certain embodiments, the immunoglobulin (Ig) CH2 district of the change in fusion rotein of the present disclosure can stem from the CH2 district of human IgG1, IgG2 or IgG4 or mouse IgG 2a (such as IGHG2c), and their sequence is set forth in the SEQ ID NO:115 of WO2011/090762,199,201 and 320 (described sequence is incorporated herein by reference).

In certain embodiments, the CH2 territory changed on position 235,318,320 and 322, has L-Ala replace (namely, there is the human IgG1 CH2 territory that L235A, E318A, K320A and K322A replace) (the SEQ ID NO:595 of WO2011/090762, described sequence is incorporated herein by reference) and the N297 that optionally exists suddenly change the human IgG1 CH2 territory of (being such as mutated into L-Ala).In some other embodiment, the CH2 territory changed on position 234,235,237,318,320 and 322, has L-Ala replace (namely, there is the human IgG1 CH2 territory that L234A, L235A, G237A, E318A, K320A and K322A replace) (the SEQ ID NO:596 of WO2011/090762, described sequence is incorporated herein by reference) and the N297 that optionally exists suddenly change the human IgG1 CH2 territory of (being such as mutated into L-Ala).

In certain embodiments, the CH2 territory of change has the human IgG1 CH2 territory that can strengthen the change of the sudden change of the immunologic competences such as such as ADCC, ADCP, CDC, complement fixation, Fc receptors bind or its any combination as known in the art.

The CH3 territory that can form the constant region for immunoglobulin of CD3 Binding peptide of the present disclosure can be the immunoglobulin (Ig) CH3 territory coming from some immunoglobulin class of various species (comprising people, mouse, rat and other Mammals) or the wild-type immunoglobulin CH3 territory of subclass (such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, IgM) or its change.In certain embodiments, CH3 territory is wild type human immunoglobulin (Ig) CH3 territory, the wild-type CH3 territory of such as human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE or IgM, respectively as described in SEQ ID NO:116,208-210,204-207 and 212 of WO2011/090762 (as described in sequence be incorporated herein by reference).In certain embodiments, CH3 territory is the wild type human IgG1CH3 territory as described in the SEQ ID NO:116 (as described in sequence be incorporated herein by reference) of WO2011/090762.In certain embodiments, CH3 territory is the human normal immunoglobulin CH3 territory changed, such as based on or stem from the CH3 territory of change in wild-type CH3 territory of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE or IgM antibody.For example, the CH3 territory of change can be the human IgG1 CH3 territory (position is numbered according to EU numerical system) on H433 and N434 of position with one or two sudden change.Sudden change on described position may participate in complement fixation.In some other embodiment, the CH3 territory of change can be human IgG1 CH3 territory, but has one or two aminoacid replacement on F405 or Y407 of position.Amino acid on described position take part in the interaction with another CH3 territory.In certain embodiments, the CH3 territory of change can be the human IgG1 CH3 territory of the change of last Methionin disappearance.The sequence in the CH3 territory of this change is set forth in (described sequence is incorporated herein by reference) in the SEQ IDNO:761 of WO2011/090762.

In certain embodiments, the CD3 Binding peptide forming polypeptide heterodimer comprises a CH3 couple, its comprise so-called " button hand-hole " sudden change (see Marvin and Zhu, ActaPharmacologica Sinica 26:649-58,2005; Ridgway etc., Protein Engineering 9:617-21,1966).More particularly, sudden change can be incorporated in each of two CH3 territories of each polypeptide chain, make CH3/CH3 associate needed for spatial complementarity force this two CH3 territories and paired with each other.For example, CH3 territory in a single chain polypeptide of polypeptide heterodimer can (" button " suddenlys change containing T366W sudden change, it uses larger aminoacid replacement p1 amino acid), and the CH3 territory in another single chain polypeptide of polypeptide heterodimer can containing Y407A sudden change (" hole " suddenlys change, and it uses the less large amino acid of aminoacid replacement).Other exemplary button hand-hole sudden change comprises the T366Y sudden change in (1) CH3 territory and the Y407T in another CH3 territory, and the T366W in (2) CH3 territories suddenlys change and T366S, L368A and Y407V sudden change in another CH3 territory.

The CH4 territory that can form the constant region for immunoglobulin of CD3 Binding peptide of the present disclosure can be the wild-type immunoglobulin CH4 territory or its immunoglobulin (Ig) CH4 territory changed that come from IgE or IgM molecule.In certain embodiments, CH4 territory is wild type human immunoglobulin (Ig) CH4 territory, the people IgE such as respectively as described in the SEQ ID NO:213 and 214 (as described in sequence be incorporated herein by reference) of WO2011/090762 and the wild-type CH4 territory of IgM molecule.In certain embodiments, CH4 territory is the human normal immunoglobulin CH4 territory changed, such as based on or stem from the CH4 territory of change in CH4 territory of people IgE or IgM molecule, described molecule has can increase or reduce the known immunocompetent sudden change relevant to IgE or IgM Fc district.

In certain embodiments, the constant region for immunoglobulin of CD3 Binding peptide of the present disclosure comprises the combination (that is, more than is selected from the constant region domains of CH2, CH3 and CH4) in CH2, CH3 or CH4 territory.For example, immunoglobulin (Ig) constant sub-region can comprise CH2 and CH3 territory or CH3 and CH4 territory.In some other embodiment, constant region for immunoglobulin can comprise two CH3 territories and not comprise CH2 or CH4 territory (that is, only two or more CH3).Formed immunoglobulin (Ig) constant sub-region multiple constant region domain can based on or stem from identical immunoglobulin molecules, or the immunoglobulin molecules of identical category or subclass.In certain embodiments, immunoglobulin (Ig) constant sub-region is IgG CH2CH3 (such as IgG1CH2CH3, IgG2CH2CH3 and IgG4CH2CH3), and can be people (such as, human IgG1, IgG2 and IgG4) CH2CH3.For example, in certain embodiments, immunoglobulin (Ig) constant sub-region comprises (1) wild type human IgG1CH2 and (namely CH3 territory, (2) have the human IgG1 CH2 of N297A replacement, CH2 (N297A)) and wild type human IgG1CH3, or the human IgG1 CH3 of the change of (3) human IgG1 CH2 (N297A) and last Methionin disappearance.

Or, multiple constant region domain can based on or stem from different immunoglobulin molecules or immunoglobulin molecules that is different classes of or subclass.For example, in certain embodiments, immunoglobulin (Ig) constant sub-region comprises people IgM CH3 territory and human IgG1 CH3 territory.The multiple constant region domain forming immunoglobulin (Ig) constant sub-region can be connected directly between together, or can be connected to each other via one or more (such as about 2-10) amino acid.

Exemplary immunoglobulin constant sub-region is set forth in the SEQ IDNO:305-309,321,323,341,342 and 762 of WO2011/090762 (described sequence is incorporated herein by reference).

In certain embodiments, the constant region for immunoglobulin of two CD3 Binding peptide chains of homodimer or heterodimer is same each other.In some other embodiment, the immunoglobulin (Ig) constant sub-region of a polypeptide chain of heterodimeric albumen is different from the immunoglobulin (Ig) constant sub-region of another polypeptide chain of described heterodimer.For example, an immunoglobulin (Ig) constant sub-region of heterodimeric albumen can containing the CH3 territory having " button " and suddenly change, and another immunoglobulin (Ig) constant sub-region of described heterodimeric albumen can containing the CH3 territory having " hole " and suddenly change.

Embodiments more of the present invention relate to the following dual specific of use or polyspecific binding molecule: the TCR mixture (such as CD3) in (i) targeted human T cell and (ii) cell surface protein, such as with redirected T cell cytotoxicity for the cell with described cell surface protein, such as the tumour cell of expressing tumor antigen so that Therapeutic cancer.

In certain embodiments, CD3 associated proteins can comprise other binding domain (such as the second binding domain) of the target of one or more combination except CD3.These other target molecules can comprise such as tumor associated antigen.In one embodiment of the invention, other binding domain one or more in conjunction with one or more following tumour antigen or with its interaction: RON, c-Met, CEACAM-6, PSMA, EpCAM, CEA, PCTA-1, STEAP-1, STEAP-2, PSCA, PSA, PAP, ALCAM (CD166), PECAM-1, EphA2, CD151, CA-125/MUC16, MUC-1, MAGE-1, TROP2, IGF1R, TGFBR2, GHRHR, GHR, IL-6R, gp130, TNFR2, OSMR β, Patched-1, Frizzled, Robo1, LT β R, CD19, CD25, CD26, CD27, CD30, CD33, CD44, CD44v6, CD63, CD80, CD81, CD86, CD100, CD151, CXCR4, CCR5, HER-2/ErbB1, HER-3/ErbB3, HER-4/ErbB4, EGFR/ErbB1, EGFRvIII isotype, MUC2, MUC3, MUC4, MUC5 _aC, MUC5 _b, MUC7, β hCG, Lewis-Y, Ganglioside, GD3,9-O-ethanoyl-GD3, GM2, GloboH, Fucose acyl GM1, Poly SA, GD2, carbonic anhydrase IX (MN/CA IX), Sonic hedgehog (Sonic Hedgehog) (Shh), Wue-1, plasma cell antigen, (film combination) IgE, melanoma chondroitin sulfate protein-polysaccharide (MCSP), CCR8, TNF-α precursor, mesothelin, A33 antigen, Ly-6, desmoglein 4, the new epi-position of E-Calcium ionorphore, fetus acetylcholine receptor, CA19-9 marker, seedling Le Shi inhibitory substance (MIS) receptor II type, sTn (sialylated Tn antigen, TAG-72), FAP (inoblast active antigen), endosialin, LG, SAS, BCMA, TWEAKR/Fn14, FGFR4, VEGFR1, VEGFR2, SSX1 and SSX2.

In certain embodiments, CD3 binding domain in conjunction with TCR binding domain T cell to be raised the target cell of expressing tumor antigen.In certain embodiments, CD3 Binding peptide can comprise the CD3 binding domain of specific binding TCR mixture or its component (such as TCR α, TCR β, CD3 γ, CD3 δ and CD3 ε) and another binding domain of specific binding tumor associated antigen.

PSMA is a kind of height-limited prostate cancer relevant cell membrane antigen.In prostate cancer cell, the expression of PSMA is 1000 times (Su etc., Cancer Res.199544:1441-1443) on normal prostatic epithelium cell.The expression of PSMA increases along with prostate cancer progress, and typically the highest in metastatic disease, hormone refractory case and higher level pathology (Israeli etc., Cancer Res.1994,54:1807-1811; Wright etc., UrologicOncology:Seminars and Original Investigations 19951:18-28; Wright etc., Urology 199648:326-332; Sweat etc., Urology 199852:637-640).In addition, PSMA be expressed in other solid tumour multiple (comprising bladder cancer, carcinoma of the pancreas, melanoma, lung cancer and kidney) galore new vessel on instead of (Chang etc., Urology 200157:801-805 on normal new vessel; Divgi etc., Clin.Cancer Res.19984:2729-3279).The binding domain of target PSMA include but not limited to described in PCT publication No. WO2012/145714 those, the mode that the document is quoted in full is incorporated herein.

In some embodiments, PSMA binding domain comprises and the aminoacid sequence being selected from following aminoacid sequence and having at least 90%, at least 95% or 100% identity: the amino acid/11-107 of (i) SEQ IDNO:212 and 124-243; (ii) amino acid/11-107 of SEQ ID NO:226 and 124-243; Or the amino acid/11-107 of (iii) SEQ ID NO:216 and 124-243.In some embodiments, protein of the present invention or molecule comprise be selected from SEQ ID NO:212,214, the aminoacid sequence of 216 and 226.PSMA binding domain that can be used according to the invention is also described in PCT publication No. WO2012/145714, include comprise WO2012/145714 aminoacid sequence SEQ ID NO:19,21,30,31, the PSMA binding domain of 34 or 35, or include the VL chain of the aminoacid sequence comprising the SEQ ID NO:5 and 23 being selected from WO2012/145714 and comprise the VH chain being selected from the SEQ IDNO:2 of WO2012/145714, the aminoacid sequence of 25 and 27.

RON (receptor tyrosine kinase, also referred to as MST1R) be the necessary receptor type protein tyrosine kinase of fetal development and also play an important role in inflammatory response (Camp etc., Ann.Surg.Oncol.12:273-281 (2005)).RON is mainly expressed in epidermal derived cell type, and propose, RON may work (Wang etc., Carcinogenesis 23:1291-1297 (2003)) as other receptor type tyrosine kinases many in Malignant Epithelium cancer progress.The activation of RON causes tumours such as involving such as cell proliferation, apoptosis suppression and cell mobility and active downstream signal conducting path occurs.Especially, RON represents epitheliomatous therapeutic targets due to process LAN in colorectal carcinoma, mammary cancer, ovarian cancer and carcinoma of the pancreas of its intracellular signaling character and/or RON.

Include but not limited to described in embodiment part those and PCT and announce those described in WO2011/090761 for the binding domain of target RON, the mode that the document is quoted in full is incorporated herein.In some embodiments, anti-RON binding domain comprises (a) VL territory, and it comprises i.CDR1 aminoacid sequence SEQ ID NO:87, CDR2 aminoacid sequence SEQ ID NO:88 and CDR3 aminoacid sequence SEQ ID NO:89; Or ii.CDR1 aminoacid sequence SEQ ID NO:90, CDR2 aminoacid sequence SEQ ID NO:91 and CDR3 aminoacid sequence SEQ ID NO:92; Or (b) VH territory, it comprises i.CDR1 aminoacid sequence SEQ ID NO:93, CDR2 aminoacid sequence SEQ ID NO:94 and CDR3 aminoacid sequence SEQ ID NO:95; Or ii.CDR1 aminoacid sequence SEQ ID NO:96, CDR2 aminoacid sequence SEQ ID NO:97 and CDR3 aminoacid sequence SEQ ID NO:98; Or the VL of (c) (a) and the VH of (b).In one embodiment, VL territory to comprise in SEQ ID NO:99 or 100 any one aminoacid sequence, and VH territory to comprise in SEQ ID NO:101,102 and 103 aminoacid sequence of any one.In another embodiment, VL and VH territory is by humanization.In certain embodiments, humanization VL to comprise in SEQ ID NO:104,105 and 106 any one aminoacid sequence, and humanization VH territory to comprise in SEQ IDNO:107-111 the aminoacid sequence of any one.In some embodiments, anti-RON binding domain includes the VL territory comprising the aminoacid sequence of any one in SEQ ID NO:99 and 100, and comprises the VH territory of the aminoacid sequence of any one in SEQ ID NO:101,102 and 103.In some embodiments, anti-RON binding domain includes the VL territory comprising the aminoacid sequence of any one in SEQ ID NO:104,105 and 106, and comprises the VH territory of the aminoacid sequence of any one in SEQ IDNO:107-111.

In some embodiments, RON binding domain comprises the aminoacid sequence with SEQ ID NO:187 with at least 90%, 92%, 94%, 96%, 97%, 98%, 99% or 100% identity and/or comprises the aminoacid sequence with SEQ ID NO:188 with at least 90%, 92%, 94%, 96%, 97%, 98%, 99% or 100% identity.In some embodiments, RON binding domain comprises (i) SEQ ID NO:187, wherein amino acid 43 (L-Ala) is through another aminoacid replacement, such as, replace through Methionin (A43K) or Threonine (A43T); (ii) SEQID NO:188, wherein amino acid 38 (glutamine) and/or 113 (glutamine) are through another aminoacid replacement, such as, replace through L-glutamic acid and/or arginine, such as Q38R and/or Q113E; Or (iii) its any combination.In some embodiments, anti-RON/ AntiCD3 McAb bispecific molecule comprise the aminoacid sequence with any one in SEQ ID NO:186,190,192 or 194 with at least 90%, 92%, 94%, 96%, 97%, 98%, 99% or 100% identity or consisting of.In some embodiments, the invention provides a kind of nucleic acid of anti-RON/ AntiCD3 McAb bispecific molecule of encoding, wherein said nucleic acid comprises the nucleotide sequence with any one in SEQ ID NO:185,189,191 or 193 with at least 90%, 92%, 94%, 96%, 97%, 98%, 99% or 100% identity.

CD19 is in B cell, and the surface comprising most of malignant B cell finds.Target CD19 can be used for suppressing and treatment B cell relative disease, leukemia and lymphoma.

In some embodiments, CD19 binding domain comprises the aminoacid sequence with the amino acid/11-111 of SEQ ID NO:196 with at least 90%, 92%, 94%, 96%, 97%, 98%, 99% or 100% identity and/or comprises the aminoacid sequence with the amino acid/11 28-251 of SEQ ID NO:196 with at least 90%, 92%, 94%, 96%, 97%, 98%, 99% or 100% identity.In some embodiments, anti-CD19/ AntiCD3 McAb bispecific molecule comprise with SEQ ID NO:196,198,200,202,204 or 206 in any one have at least 90%, 92%, 94%, 96%, 97%, 98%, 99% or 100% identity aminoacid sequence or consisting of.In some embodiments, the invention provides a kind of nucleic acid of anti-CD19/ AntiCD3 McAb bispecific molecule of encoding, wherein said nucleic acid comprises the nucleotide sequence with any one in SEQ IDNO:195,197,199,201,203 or 206 with at least 90%, 92%, 94%, 96%, 97%, 98%, 99% or 100% identity.

HER2 is also referred to as people ErbB2.The process LAN (often but not always due to gene amplification) of HER2 is also been observed in mammary cancer, ovarian cancer and other cancer knurl (comprising cancer of the stomach, carcinoma of endometrium, salivary-gland carcinoma, lung cancer, kidney, colorectal carcinoma, thyroid carcinoma, carcinoma of the pancreas and bladder cancer).The binding domain of target HER2 include but not limited to that PCT announces described in WO2009/055074 those, the mode that the document is quoted in full is incorporated herein.

In some embodiments, CD3 Binding peptide comprises the aminoacid sequence with SEQ ID NO:57,59,61,63,65,67,69,71,186,190,192,194,196,198,200,202,204 or 206 with at least 90%, at least 95% or 100% identity.

In some embodiments, CD3 Binding peptide is a part for heterodimer.In some embodiments, heterodimer comprises a pair single chain polypeptide, and wherein said strand is to the aminoacid sequence comprised be selected from following pairing and have at least 90%, at least 95% or 100% identity: SEQ ID NO:210 and 247, SEQ ID NO:210 and 218, SEQ ID NO:210 and 220, SEQ ID NO:208 and 249, SEQ ID NO:208 and 222 or SEQ IDNO:208 and 224, SEQ ID NO:212 and 218, SEQ ID NO:216 and 222, SEQID NO:228 and 226 or SEQ ID NO:214 and 218.

The present invention also comprises coding CD3 Binding peptide as described in this article, or the nucleic acid (such as DNA or RNA) of dimerization as described in this article or the protein-bonded one or more polypeptide chain of heterodimeric CD3.Nucleic acid of the present invention comprise have with as in SEQ ID NO:5,7,9,11,13,15,17,19,27,29,31,37 and 39 the substantially same region of the polynucleotide that provides nucleic acid or comprise the nucleic acid of coding region of same or similar polypeptide of encoding.In certain embodiments, according to nucleic acid of the present invention with as in SEQ ID NO:5,7,9,11,13,15,17,19,27,29,31,37 and 39 the peptide coding polynucleotide that provides have at least 80%, typically at least about 90% and more typically at least about 95% or at least about 98% identity.Nucleic acid of the present invention also comprises complementary nucleic acid.In some cases, sequence when comparison by complete complementary (without mispairing).Under other circumstances, about 20% mispairing at most may be there is in sequence.Provide the nucleic acid of protein-bonded both first and second polypeptide chains of coding heterodimeric CD3 of the present invention in some embodiments of the present invention.Nucleotide sequence provided herein can access to your password the expression that son optimization, degenerate sequence, silent mutation and other DNA technique are used to optimize in specific host, and described sequence modification is contained in the present invention.

In the carrier the polynucleotide molecule comprising wanted polynucleotide sequence is bred by described molecule is put.The present invention also comprises containing nucleic acid of the present invention and/or the expression vector of expressing polypeptide of the present invention.Use virus and non-virus carrier, comprise plasmid.The selection of plasmid will depend on cell type and propagation object that needs are bred.Some carrier is applicable to amplification and manufactures a large amount of required DNA sequence dnas.Other carrier is suitable for expressing in the cell in cultivation.Other carrier is suitable for carrying out shifting and expressing in the cell of whole animal or human.The selection of suitable carrier is completely within the control of those skilled in the art.Many such carriers can derive from market.Typically be connected to the restriction enzyme sites of cracking in carrier by means of DNA ligase by part or full length polynucleotide insertion vector.Or, desired nucleotide sequence can be inserted by In vivo homologous recombination.Typically, this be attended by homology region to be connected in carrier want on the both sides of nucleotide sequence.For example, to be connected by oligonucleotide or by polymerase chain reaction, use comprise homology region and want the primer of a part for nucleotide sequence to add homology region.

For expression, expression cassette or system can be adopted.In order to express the nucleic acid of coding polypeptide disclosed herein, be incorporated in host cell by the nucleic acid molecule of coding said polypeptide, described nucleic acid molecule is operably connected to the adjustment sequence of the transcriptional expression that can control in expression vector.Present invention comprises and comprise nucleic acid of the present invention and/or expression vector, such as the host cell of the nucleic acid of code book invention polypeptide.Except the such as transcriptional regulatory sequences such as promotor and enhanser, expression vector can comprise translational regulation sequence and be suitable for the marker gene of the cell selecting to carry expression vector.Gene product expression coded by polynucleotide of the present invention, in any suitable expression system, comprises such as bacterium, yeast, insect, Amphibians and mammlian system.In expression vector, peptide coding polynucleotide is connected to regulates sequence to obtain desired expression character in due course.These can comprise promotor, enhanser, terminator, operon, suppression and inductor.Promotor can be (such as the coming from the promotor of CMV, SV40, elongation factor or LTR sequence) of modulability (such as coming from the promotor of steroid inducibility pIND carrier (Invitrogen)) or composition.Use as these being connected to desired nucleotide sequence about the technology be connected to as described in carrier above.Any technology as known in the art can be used.Therefore, expression vector generally will provide and transcribe and Translation initiator, and it can be epigamic or composition, wherein transcription initiation region and transcribe with translate end district transcribe control under to be operably connected coding region.

Can expression cassette (" expression unit ") be incorporated in variety carrier, such as plasmid, BAC, YAC, phage (such as λ, P1, M13 etc.), plant or animal virus vector are (such as, based on retroviral carrier, adenovirus carrier) etc., wherein carrier is normally to select to comprise the ability of the cell of expression vector for feature.Carrier can provide karyomit(e) to maintain outward, particularly as plasmid or virus, or for being incorporated in host chromosome.When needs karyomit(e) maintains, provide origin sequences so that plasmid replication outward, it can be low or high copy number.Multiple marker can be used for selecting, and exactly prevents toxin, more precisely prevents those markers antibiotic.Select selected particular marker according to the character of host, wherein in some cases, can supplement with auxotroph host.The introducing of DNA construct can use any suitable method, comprises the DNA of such as combination, Bacterial Transformation, calcium deposit, electroporation, fusion, transfection, uses viral vector infection, biological bullet method etc.

Therefore, the protein used in the present invention can produce in genetic engineering modified host cell according to routine techniques.Suitable host cell is can transform with exogenous DNA or transfection and the cell type grown in the medium, and comprise the higher eukaryotes (comprising the culturing cell of multicellular organisms) of bacterium, fungal cell and cultivation, the mammalian cell particularly cultivated.Disclose the DNA molecular for manipulating clone with Publication about Document and exogenous DNA is incorporated into the technology in multiple host cell: Sambrook and Russell, MolecularCloning:A Laboratory Manual (the 3rd edition, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, NY, 2001); With Ausubel etc., Short Protocols inMolecular Biology (the 4th edition, John Wiley & Sons, 1999).

For example, in order to the recombinant expressed homologous dimerization CD3 associated proteins comprising two same CD3 Binding peptide chains as described in this article, expression vector generally will comprise the nucleic acid segment of the coding CD3 Binding peptide being operably connected to promotor.In order to the recombinant expressed heterodimeric CD3 associated proteins comprising the first and second different polypeptide chains, the first and second polypeptide chains can by carrier coexpression independent in host cell to express whole heterodimeric albumen.Or in order to expressing heterologous dimerization CD3 associated proteins, the first and second polypeptide chains can by expression unit coexpression independent in the identical carrier in host cell to express whole heterodimeric albumen.By routine techniques, expression vector is transferred to host cell, then cultivated through the cell of transfection to produce coded polypeptide by routine techniques, thus produce the CD3 associated proteins of correspondence.

In order to be directed to by recombinant protein in the secretion path of host cell, in expression vector, provide secretory signal sequence (also referred to as leader sequence).Secretory signal sequence can be the secretory signal sequence of the natural form of recombinant protein, maybe can stem from another secretory protein or again synthesize.Secretory signal sequence is operably connected to the DNA sequence dna of coded polypeptide, that is, two sequences is engaged in correct reading frame and position the polypeptide newly synthesized is directed in the secretion path of host cell.Secretory signal sequence usually relative to coding related polypeptide DNA sequence dna be positioned at 5', but some signal sequence can be arranged in associated dna sequence other place (see such as Welch etc., U.S. Patent number 5,037,743; Holland etc., U.S. Patent number 5,143,830).In particular variant, secretory signal sequence used according to the invention has aminoacid sequence MEAPAQLLFLLLLWLPDTTG (SEQ IDNO:79).

The mammalian cell cultivated is the host being suitable for producing the recombinant protein used in the present invention.Method for being incorporated in mammalian host cell by exogenous DNA comprises transfection (Wigler etc., Cell 14:725,1978 of calcium phosphate mediation; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981; Graham and Van der Eb, Virology52:456,1973), electroporation (Neumann etc., EMBO J.1:841-845,1982), the transfection (Ausubel etc. of DEAE dextran mediation, the same) and liposome-mediated transfection (Hawley-Nelson etc., Focus 15:73,1993; Ciccarone etc., Focus 15:80,1993).For example, in the mammalian cell cultivated, recombinant polypeptide is produced to disclose in Publication about Document: Levinson etc., U.S. Patent number 4,713,339; Hagen etc., U.S. Patent number 4,784,950; Palmiter etc., U.S. Patent number 4,579,821; And Ringold, U.S. Patent number 4,656,134.The example of suitable mammalian host cell comprises African green monkey kidney cell (Vero; ATCC CRL 1587), HEKC (293-HEK; ATCC CRL 1573), baby hamster kidney cell (BHK-21, BHK-570; ATCC CRL 8544, ATCC CRL10314), Madin-Darby canine kidney(cell line) (MDCK; ATCC CCL 34), Chinese hamster ovary cell (CHO-K1; ATCC CCL61; CHO DG44; CHO DXB11 (Hyclone, Logan, UT); Also see such as Chasin etc., Som.Cell.Molec.Genet.12:555,1986)), rat pituitary cell (GH1; ATCC CCL82), HeLa S3 cell (ATCC CCL2.2), rat hepatocytes cancer cells (H-4-II-E; ATCC CRL 1548), through SV40 transform monkey-kidney cells (COS-1; ATCC CRL 1650) and mouse protoblast (NIH-3T3; ATCC CRL1658).Other suitable clone is well known in the art, and can derive from the public preservation centers such as the American type culture collection of such as Manassas, Virginia.Strong transcripting promoter can be used, be such as obtained from the promotor of SV-40 or cytomegalovirus.See such as U.S. Patent number 4,956,288.Other suitable promotor comprises the promotor (U.S. Patent number 4,579,821 and 4,601,978) and adenovirus major late promoter that come from metallothionein gene.

Medicament selection is generally used for the mammalian cell selecting the cultivation of wherein having inserted foreign DNA.Described cell is commonly referred to " transgenic cell ".To cultivate under selective agent exists and the cell that genes involved can be passed to its offspring is called " stablizing transgenic cell ".Exemplary selectable markers thing comprises encoding gene antibiotic neomycin to resistance, and it allows to select under the Liu Suanyan NEOMYCIN SULPHATE types of drug such as such as G-418 exist; For the gpt gene of xanthine-guanine phosphoribosyl transferase, it allows host cell growth under mycophenolic acid/xanthine exists; With the marker of the resistance provided for bleomycin, bleomycin, miewensu and Totomycin (see such as Gatignol etc., Mol.Gen.Genet.207:342,1987; Drocourt etc., Nucl.Acids Res.18:4009,1990).Selective system can also for increasing the expression level of genes involved, and this process is called " amplification ".Under existing at low-level selective agent, cultivate transgenic cell, then increase the amount of selective agent until select can produce introduce the high-level product of gene cell increase.The exemplary selectable marker that increases is Tetrahydrofolate dehydrogenase, and it can give the resistance for methotrexate.Other drug resistance gene (such as hygromycin resistance, multi-drug resistance, puromycin acetyltransferase) can also be used.

Other higher eukaryotes can also be used as host, comprise insect cell, vegetable cell and avian cells.Sinkar etc., J.Biosci. (Bangalore) 11:47-58,1987 have looked back the carrier of use Agrobacterium rhizogenes as expressing gene in vegetable cell.To disclose Transformation of insect cells in Publication about Document and to produce extraneous polypeptide wherein: Guarino etc., U.S. Patent number 5,162,222; WO 94/06463 is announced with WIPO.

Can with the recombinate shape virus infection insect cell usually stemming from autographa californica nuclear polyhedrosis virus (AcNPV).See King and Possee, The BaculovirusExpression System:A Laboratory Guide (Chapman & Hall, London); O'Reilly etc., Baculovirus Expression Vectors:A Laboratory Manual (Oxford University Press., New York 1994); With Baculovirus ExpressionProtocols.Methods in Molecular Biology (Richardson compiles, Humana Press, Totowa, NJ, 1995).Recombinant baculovirus can also be produced by the system based on transposon using (J.Virol.67:4566-4579,1993) such as Luckow to describe.This system utilizes transfer vector, can derive from market (BAC-TO-BAC test kit in kit form; LifeTechnologies, Gaithersburg, MD).Transfer vector (such as PFASTBAC1; LifeTechnologies) containing the Tn7 transposon for the DNA of encoding related proteins matter being moved in Large plasmid in Baculovirus Gene group that (being called " rod granule ") form is maintained in intestinal bacteria.See Hill-Perkins and Possee, J.Gen.Virol.71:971-976,1990; Bonning etc., J.Gen.Virol.75:1551-1556,1994; With Chazenbalk and Rapoport, J.Biol.Chem.270:1543-1549,1995.In addition, transfer vector can comprise and to extend with coded polypeptide or the DNA of avidity label carries out frame endomixis as disclosed above.Use technology as known in the art, the transfer vector containing protein coding DNA sequences is transformed in e. coli host cell, and screen described cell for the rod granule of the interruption lacZ gene containing instruction recombinant baculovirus.Common technology is used to be separated containing the genomic bacmid dna of recombinant baculovirus, and for transfection bomyx mori cell, such as Sf9 cell.Produce the recombinant virus of expressing related protein subsequently.Recombinant virus storing solution is manufactured by method usually used in this field.

Protein is produced, uses recombinant virus infection host cell, typically be stem from meadow mythimna separata, noctuid is coveted on meadow (such as Sf9 or Sf21 cell) or cabbage looper (such as HIGH FIVE cell; Invitrogen, Carlsbad, CA) clone.General see Glick and Pasternak, Molecular Biotechnology, Principles & Applications ofRecombinant DNA (ASM Press, Washington, D.C., 1994).Also see U.S. Patent number 5,300,435.Serum free medium is used to grow and maintain described cell.Suitable substratum preparation is well known in the art and can available from supplier on the market.Make described cell from about 2-5 × 10 ⁵the inoculum density of individual cell grows into 1-2 × 10 ⁶the density of individual cell, now adding infection multiplicity (MOI) is 0.1 to 10, is more typically the recombinant virus storing solution close to 3.The program general description used is (see such as King and Possee, the same in available laboratory manual; O'Reilly etc., the same; Richardson, the same).

Also can use fungal cell in the present invention, comprise yeast cell.Yeast specie relevant especially in this respect comprises yeast saccharomyces cerevisiae, Pichia pastoris and pichia methanolica.For example, disclose by exogenous DNA transformed saccharomyces cerevisiae cell and the method being produced recombinant polypeptide by it with Publication about Document: Kawasaki, U.S. Patent number 4,599,311; Kawasaki etc., U.S. Patent number 4,931,373; Brake, U.S. Patent number 4,870,008; Welch etc., U.S. Patent number 5,037,743; With Murray etc., U.S. Patent number 4,845,075.By by the cell there is phenotype that resistance or the selectable marker that can grow when there is not specific nutrition (such as leucine) determine usually selecting through transforming.Exemplary vector systems for yeast saccharomyces cerevisiae is the POT1 carrier system disclosed in (U.S. Patent number 4,931,373) such as Kawasaki, and it allows by growing the cell selected through transforming in containing the substratum of glucose.For the suitable promotor of yeast and terminator comprise come from glycolytic enzyme gene (see such as Kawasaki, U.S. Patent number 4,599,311; Kingsman etc., U.S. Patent number 4,615,974; And Bitter, U.S. Patent number 4,977,092) and those of alcohol dehydrogenase gene.Also see U.S. Patent number 4,990,446,5,063,154,5,139,936 and 4,661,454.For other yeast, the conversion system comprising multiple-shaped nuohan inferior yeast, schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis (D C.) Corola., Pichia pastoris, pichia methanolica, Pichia guilliermondii and Candida maltosa is well known in the art.See such as Gleeson etc., J.Gen.Microbiol.132:3459-3465,1986; Cregg, U.S. Patent number 4,882,279; With Raymond etc., Yeast 14:11-23,1998.Can according to McKnight etc., U.S. Patent number 4,935, the method for 349 utilizes Aspergillus cell.Sumino etc., U.S. Patent number 5,162,228 disclose for transforming the method for producing yellow cephalo.Lambowitz, U.S. Patent number 4,486,533 disclose the method for transforming neurospora.U.S. Patent number 5,716,808,5,736,383,5,854,039 and 5,888, disclose in 768 and produce recombinant protein in pichia methanolica.

Prokaryotic host cell, comprising the bacterial strain of bacteria Escherichia coli, tooth born of the same parents Bacillaceae and other Pseudomonas, is also host cell available in the present invention.Well-known (see such as Sambrook and Russell, the same) in the art for transforming these hosts and cloning by expression in the technology of foreign DNA sequence wherein.When expressing recombinant protein in the bacteriums such as such as intestinal bacteria, albumen can be retained in tenuigenin, typically in insoluble particle form, or can be directed to periplasmic space by bacterial secretion se.In the previous case, cell is dissolved, and particle is recovered and uses such as guanidinium isothiocyanate or urea to make its sex change.Then, metaprotein can refolding and by dilution denaturing agent carry out dimerization, such as by dialysing for the combination of urea soln and Glutathione reduced and oxidized, dialyse for buffered saline solution subsequently.In replacement scheme, protein can reclaim in solubilized form and be separated when not using denaturing agent from tenuigenin.Protein reclaims from cell in aqueous extract form in such as phosphate buffered saline.In order to capture related protein, extract is directly applied to chromatographic media, such as fixing antibody or heparin-sepharose post.Can by destroying cell (such as, by supersound process or osmotic shock) to discharge the inclusion of periplasmic space and to reclaim protein, thus avoid the needs of sex change and refolding and reclaim in solubilized and the secretory protein of functional form from periplasmic space.Antibody can be produced according to known method in bacterial host cell, comprise single-chain antibody.See such as Bird etc., Science242:423-428,1988; Huston etc., Proc.Natl.Acad.Sci.USA 85:5879-5883,1988; With Pantoliano etc., Biochem.30:10117-10125,1991.

In the substratum containing other component needed for nutritious and selected host cell growth, the host cell through conversion or transfection is cultivated according to conventional procedure.Multiple suitable substratum, comprising defined medium and compound substratum, is as known in the art, and generally comprises carbon source, nitrogenous source, indispensable amino acid, VITAMIN and mineral substance.Substratum optionally can also contain the such as component such as somatomedin or serum.Generally will lack by such as medicament selection or essential nutrients the growth medium selecting the cell be used for containing external source interpolation DNA, described essential nutrients be by expression vector carries or the selectable marker be jointly transfected in host cell supplement.

Can utilize conventional protein purification method, the combination typically via chromatographic technique carrys out purifying CD3 associated proteins.General see Affinity Chromatography:Principles & Methods (Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988); Scopes, Protein Purification:Principles and Practice (Springer-Verlag, New York 1994).The protein that purifying comprises immunoglobulin fc region can be carried out by carrying out affinity chromatography on fixing albumin A or Protein G.Other purification steps such as such as gel-filtration can be used to obtain desired purity level or to carry out desalination, buffer-exchanged etc.

Present invention includes the composition comprising CD3 Binding peptide of the present invention and pharmaceutically acceptable supporting agent, thinner and/or vehicle.

In one embodiment of the invention, monospecific CD3 Binding peptide is administered to the patient suffering from the autoimmune disorderss such as such as arthritis deformans.In another embodiment of the invention, monospecific CD3 Binding peptide of the present invention is administered to the experimenter that will carry out organ transplantation.

In yet another aspect, the invention provides a kind of method being used for the treatment of the illness that such as cancer etc. is feature with tumour antigen process LAN.In general, the comprising as described in this article of experimenter's administering therapeutic significant quantity that described method comprises to the described treatment of needs can in conjunction with the CD3 associated proteins of the second binding domain of tumour antigen.In some embodiments, the induction of CD3 associated proteins is redirected the cell of T cell cytotoxicity (RTCC) for expressing tumor antigen in experimenter.

In some change programme of described method, described illness is cancer.In other change programme, described illness is autoimmune disorders.Present invention also offers the method being used for the treatment of cancer or autoimmune disorder, comprise the composition described herein to patient therapeuticallv's significant quantity in need or CD3 Binding peptide.

In each embodiment of methods for the treatment of described in this article, send CD3 associated proteins in the mode meeting the ordinary method relevant to the disposal of the disease sought treatment or illness.According to disclosure herein, the CD3 associated proteins of significant quantity be administered to time of needing the experimenter of this treatment to continue to be enough to prevent or treat described disease or illness or use under the condition being enough to prevent or treat described disease or illness.

In order to use, CD3 associated proteins is formulated as pharmaceutical composition.Comprise the composition that the protein-bonded pharmaceutical composition of CD3 can prepare to prepare according to known method pharmaceutically useful, wherein combined therapy molecule, in the form with the mixture of pharmaceutically acceptable supporting agent.If using of composition can be tolerated by subject patient, be then referred to as " pharmaceutically acceptable supporting agent ".Sterile phosphate buffer physiological saline is an example of pharmaceutically acceptable supporting agent.Other suitable supporting agent is well-known for those skilled in the art.(see such as Gennaro (volume), Remington's Pharmaceutical Sciences (Mack PublishingCompany, the 19th edition, 1995).) preparation can also comprise one or more vehicle, sanitas, solubilizing agent, buffer reagent, albumin, to prevent the protein losses etc. on bottle surface.

The protein-bonded pharmaceutical composition of CD3 will be comprised and be administered to experimenter to treat significant quantity.According to method of the present invention, CD3 associated proteins can be administered to experimenter by multiple mode of administration, comprises such as by intramuscular, subcutaneous, intravenously, atrium, in intraarticular, parenteral, nose, in lung, in skin, pleura, in sheath and oral administration path.For prevention and therapy object, be administered to experimenter and such as can send mode, via sending continuously in time expand section (such as continuously dermal delivery) or with repetitive administration scheme (such as, in per hour, every day or weekly) with single bolus.

" treatment significant quantity (or dosage) " or " significant quantity (or dosage) " of molecule or composition is the amount that can produce statistically evident effect (such as statistically slow down progression of disease significantly or statistically improve organ dysfunction significantly) in one or more symptoms alleviating illness.Precise dosage by clinicist according to recognised standard, will consider that the character of the symptom for treating and severity, Patient Characteristics etc. determine.Dosage fixes in the level of those skilled in the art really.When mentioning combination, treatment effective dose refers to the combined amount of the activeconstituents producing result for the treatment of, no matter is use successively or use simultaneously (such as, using in same preparation or in independent preparation simultaneously).

The decision of effective dose in this article typically based on Research of Animal Model for Study and people's clinical trial subsequently, and by determining that can significantly reduce the generation of experimenter's illness or the effective dose of severity and application program in model subject is instructed.The effective dose of the present composition depends on many different factors and changes, comprise the means of using, target site, patient physiological condition, patient are people or animal, the other medicines process of using, treatment are preventative or curative, and the given activity of composition itself draws with it ability that will react in individuality.Usually, patient is people, but in some diseases, patient can be non-human mammal.Typically, regulate dosage regimen to provide optimum therapeutic response, such as, to optimize security and effect.

Embodiment

embodiment 1: design and build the AntiCD3 McAb SMIP molecule that theoretical pI reduces

L1, L2, H1, H2 and H3 (see Fig. 3 and 4) are heavy chain and the light chain of the humanization Cris-7 previously built, and analyze for combination and activity.H3L1 is used to manufacture SMIPDRA209 (SEQ ID NO:3 and 4; IgG4AA ADCC-CDC null Fc) and SMIPDRA161 (IgG4AA N297A Fc).Although these constructs compare with other AntiCD3 McAb therapeutical agent of the prior art the feature presenting improvement with OKT3, but when being expressed in (see Fig. 2 A and 2B) in Chinese hamster ovary celI, compared with other SMIP molecule, described construct shows significantly shorter transformation period, higher theoretical pI (see Fig. 1) and is in " montage " of binding domain or front hinge area.

Design AntiCD3 McAb pI variant is to reduce the pI of binding domain and/or front hinge area.In order to manufacture AntiCD3 McAb pI variant, by Blue Heron, new heavy chain and light chain (L3, L4, H4, H5 and H6) being sorted and being synthesized.See table 3 and Fig. 3 and 4.

Table 3:CD3 variable heavy chain and light chain

H4 and H3 compares has 2 point mutation.Positive charge residue (K becomes Q) has been replaced in a sudden change, and another sudden change introduces negative charge (Q becomes E).Two kinds change be all intended to reduce protein pI (or by remove positive charge, or by introduce negative charge).Frame sequence carries out suddenly change to avoid losing avidity, and the new residue empirical tests introduced is present in the position can avoiding immunogenicity risk in germline sequence.By using germline sequence as homing sequence to reduce the pI of humanized binding domain, aminoacid replacement can be carried out to add ubiquitous amino acid in germline sequence.These amino acid can same position in germline sequence or close position.See Fig. 5.

H5 is designed by carrying out " again humanization " (as compared to DRA209 with DRA161) in the mode producing the pI lower than DRA209 with DRA161 to sequence of heavy chain.This is undertaken by the heavy chain that design is suitable with the VH1 framework of IGHV1-3*01 heavy chain germline sequence.

H6 is designed by carrying out " again humanization " (as compared to DRA209 with DRA161) in the mode producing the pI lower than DRA209 with DRA161 to sequence of heavy chain.In other words, be germline sequence instead of previous humanized sequence for designing the homing sequence of H6.Specifically, this is by using the VH3 frame design heavy chain of IGHV3-21*01 sequence of heavy chain to carry out.

Compared with L1, L3 has 6 point mutation, wherein make RW suddenly change (that is, replace positive charge residue, R becomes L) become LL (using LL to be because LL is more common than LW in germline sequence), and with Jk4 sequence (VEIK; SEQ ID NO:253) replace a part (LQIT in J κ (Jk) district; SEQ ID NO:252).Jk4 is the Jk district of the better coupling based on Cris-7H_CDR3 sequence.See Fig. 6.Again illustrate, only suddenly change in framework region, and the new residue empirical tests introduced is present in the position can avoiding immunogenicity risk in germline sequence.

L4 is designed by carrying out " again humanization " (as compared to DRA209 with DRA161) in the mode producing the pI lower than DRA209 with DRA161 to sequence of light chain.The framework of the Vk3 of IGKV3-11*01 sequence of light chain is used to carry out humanization again as homing sequence to L4.

Based on Theoretical Calculation, expect that all new light chains and heavy chain all have lower pI.Carrier NTI (Invitrogen) is used to help these sequences to be put together (connecting these sequences).Germline sequence is available from NCBI website (http://www.ncbi.nlm.nih.gov/igblast/).

Build in the front hinge area of otherwise same with DRA209 and DRA161 SMIP molecule and comprise the amino acid whose variant be substituted.Front hinge area is the short amino acid sequence connecting binding domain and hinge area, such as, can produce by interpolation restriction site to coding nucleotide sequence.

Manufactured a kind of variant relates to the RRT sequence of being suddenlyd change in the front hinge area of generation SSS displacement DRA209 by PCR.Do the residue (arginine) removing two positively chargeds so intentionally.Experiment display, the pI that this change is translated into protein reduces.This construct still has H3L1 combination and gives its newname DRA219.Except removing the residue (two arginine) of positively charged, this SSS sudden change also introduces the Xhol restriction site of the pI variant construct allowing simple assembling newer.

Use standard molecular biological technique, use identical Fc afterbody as DRA209, use the following combination of H and L to build SMIP construct: a) H4L1 (has IgG4AAADCC-CDC null FC; In this article also referred to as DRA222; SEQ ID NO:7 and 8), b) H4L3 (there is IgG4AA ADCC-CDC null Fc; In this article also referred to as DRA221; SEQ ID NO:9 and 10), c) H3L3 (there is IgG4AA ADCC-CDCnull Fc; In this article also referred to as DRA223; SEQ ID NO:11 and 12), d) H5L4 (there is IgG4AA ADCC-CDC null Fc; In this article also referred to as DRA224; SEQ IDNO:13 and 14) and e) H6L4 (there is IgG4AA ADCC-CDC null Fc; In this article also referred to as DRA225; SEQ ID NO:15 and 16).See Fig. 7 A and 7B.

All these constructs are transfected in HEK cell to produce protein for use in external binding.All constructs all express wanted protein, and except DRA225 construct, it does not have obvious expression.Find other constructs all (i) there is the pI lower than parent molecule DRA209 in conjunction with CD3 (DRA222 has best combination to a certain extent) and (ii) in various degree.Measure pI can obtain in table 4.

Table 4: measure pI

Construct	Measure pl
		DRA209	9.0
DRA219	8.4
		DRA221	8.2
DRA222	7.5
		DRA223	7.2
DRA224	6.8

Lymphocyte reaction (the MLR that DRA219, DRA221, DRA222, DRA223 and DRA224 suppress people to mix; Data are not shown), and the amount suppressed is relevant to its measured CD3 binding ability.

DRA219 (H3L1IgG4AA ADCC-CDC null FC; SEQ ID NO:5 and 6) and DRA222 (SEQ ID NO:7 and 8) all keep in conjunction with CD3 and there is lower measurement pI, and it is cloned into further to produce two constructs again in the pEE12.4 carrier (IgG4AAN297A) of the different Fc afterbody of coding, be DRA233 (H3L1 respectively; SEQ IDNO:17 and 18) and DRA234 (H4L1; SEQ ID NO:19 and 20).In addition, DRA219 and DRA222 is cloned into express in CHO in pEE12.4 carrier, and difference called after DRA228 and DRA229.In fact, DRA219 is equivalent to DRA228 and DRA222 is equivalent to DRA229.All constructs (DRA228, DRA229, DRA233 and DRA234) are all transfected in Chinese hamster ovary celI subsequently and study for binding, protein characterization and PK to produce protein.See Fig. 8.

By albumin A affinity chromatography, AKTA purifier HPLC system is used to be purified into protein from cell conditioned medium liquid.By pre-fill POROS A post (Life Technologies) for preparation in vitro, and by pre-fill MabSuRe albumin A post (GE Healthcare) for material in body.Load sample and use citrate buffer elution under PBS exists.With in TRIS damping fluid and eluant.

Beckman PA 800 instrument carries out capillary isoelectric focusing and iso-electric point (pI) for measuring protein purification.Analyze according to standard Beckman cIEF scheme.In the solution of 3M urea in clEF gel (Beckman), prepare sample, this solution is supplemented with Pharmalyte 3-10 amphotericeledrolyte (GE Healthcare), negative electrode and anodic stabilization agent and synthetic peptide pI marker (Beckman).Sample is incorporated in neutral kapillary (Beckman), then by capillary inlet to be immersed in anolyte, outlet to be immersed in catholyte and within 15 minutes, to focus on kapillary application 25kV potential duration.After focusing, by chemical means make sample flow with make separative component all pass through the fixed wave length detector of the specific absorption measured under 280nm.According to the iso-electric point (pI) of the typical curve calculating all samples that the result by synthetic peptide pI marker builds.See Fig. 9-11.Construct has following measurement pI:DRA228=8.4; DRA229=7.5; DRA233=8.6; DRA234=8.0, and DRA161=9.2 and DRA209=8.8.Therefore, all pI variants of expression all have the pI lower than parent construct DRA161 or DRA209 of its correspondence.

embodiment 2: AntiCD3 McAb sMIP molecule is in conjunction with T cell

Use that standard density gradient is centrifugal from fresh whole blood, be separated people's peripheral blood mononuclear cell (PBMC), and washing three times in containing the PBS of 0.2%BSA.By cell with 200,000 PBMC/ hole to be seeded in 96 hole U-shaped base plates and to use on ice 50ul antibody with nM concentration markers 30 minutes as indicated in fig. 12.Plate washed three times and mark 30 minutes with the 50ul antibody mixed solution that the anti-CD5-APC containing optimum concn adds F (ab') 2 goat anti-hu IgG Fc-PE.Plate washed three times and fixedly spend the night under 4C in the PBS of 120ul containing 1% polyoxymethylene.All antibody incubations and washing are all carry out with cold PBS, 0.2%BSA, 2nM EDTA on ice.By flow cytometry, use BD LSRII analyzer analytic sample.60 microlitres are collected from each hole.Use Flowjo software, select T cell (FCS is to SSC, CD5+ cell) to analyze collected sample by lock.Average fluorescent strength (MFI) on PE passage is shown in figure.DRA227SMIP only with comparing and not containing CD3 or T cell binding domain, and does not carry out the modification for reducing pI.See Figure 12.All retain binding activities in conjunction with all test pI variants of CD3 and DRA228 and DRA229, be very similar to their female construct.

embodiment 3: the AntiCD3 McAb with the pI of reduction sMIP molecule is shown in Chinese hamster ovary celI reach better

size Exclusion Chromatography: use the high molecular of Size Exclusion Chromatography assessment purified protein to assemble thing content.HPLC system is equipped with Agilent 1200 series that quaternary pump, solvent degas device, DAD and temperature control self-actuated sampler.The Tosoh TSKgelG3000SWxl post being used in balance in the running damping fluid (pH 7.2) of 200mM potassiumphosphate and 250mM Repone K carries out chromatographic separation.Solvent flow rate is 1mL/ minute and sample implantation quality is 50 μ g.Effluent is monitored at a wavelength of 280 nm with Agilent DAD detector.

sDS-PAGE: use sex change SDS-PAGE (SDS-PAGE) to assess the integrity of purified protein.Prepare sample with Novex sample buffer (Invitrogen), and before being loaded in 4-20%Novex Tris-glycine gels (Invitrogen), at 85 DEG C, heat 3 minutes to guarantee complete sex change.Before heating steps, optionally by interpolation Novex reductive agent also raw sample.Continue within 110 minutes, to make protein circulate and be separated by applying 125V in the Electrophoresis Lab being filled with Tris-glycine buffer (Invitrogen comes from 10X storing solution) on gelifying agent.By carrying out observing protein bands of a spectrum with SimplyBlue Safe Stain (Invitrogen) dyeing.See Figure 13-14.Test pI variant shows the montage reduced to some extent compared with its parent molecule DRA161.

cE-SDS: on Beckman PA 800 instrument, carry out the capillary electrophoresis in SDS, and be used for carrying out quantitatively the lower molecular weight montage content of purified protein.The sample of preparation 1mg/mL concentration in 50%SDS sample buffer (Beckman), and before analysis, at 85 DEG C, heat 3 minutes to guarantee complete sex change.By electronic for sample (10kV, 20s) be injected into and be filled with in the naked fused silica kapillary of sds gel damping fluid (Beckman), and by within 30 minutes, being separated at kapillary (reversed polarity) upper application 15kV voltage keeps.Detector window is measured the specific absorption under 220nm and 280nm.See Figure 15-16.The quality of this display pI variant proteins is better than parent molecule, such as, due to lower apparent proteolysis, is sometimes referred to as in this article " montage ".

embodiment 4: AntiCD3 McAb SMIP pI Variant molecules represents the transformation period of increase

The time 0, female BAl BIc/c mouse is injected to the 200 μ L PBS containing 200 μ gDRA234 or DRA233 through intravenously (IV).Inject often organizing three mouse at each time point.After injection, draw blood via the mouse of cardiac puncture to anesthesia at indicated time point, and collect serum as described below.Enzyme-linked immunosorbent assay (ELISA) is used to measure the serum-concentration of DRA234 and DRA233.By non-segmentation analysis, use WinNonlin ^tMprofessional software (v5.3) and application use with the precompile model 201 of sparse sampling for IV bolus to estimate the pharmacokinetics configuration parameter of each protein.

At time 0 point, mouse is contained to the 200 μ L PBS of 200 μ g (about 10mg/kg) DRA233 or DRA234 through intravenous injection.Inject often organizing three mouse at each time point.Via cardiac puncture to anesthesia mouse draw blood, and after injection t=15 minute and 2,6,24,48,72,96,168,336 and 504 hr collections serum.Specific sandwich ELISA is used to measure the serum-concentration of each protein.Results expression is for change average serum concentration (μ g/mL) ± SD in time.With linear (A) and logarithm (B) form display data.See Figure 17.

Sandwich ELISA is used to detect anti-drug antibodies (ADA), wherein DRA161 (primitive form of DRA233 and DRA234) is coated with onboard to capture ADA, and anti-mouse IgG (H & L) HRP is for detecting the ADA of combination.Use peroxidase substrate to measure the mixture combined, fluorescence plate reader reads result.Results expression is that mean fluorecence unit (FU) is relative to serum dilution.For DRA233, the ADA level at 504 hours places is clearly visible, and possible low-level originates in 336 hours; But all ADA is there is not in any DRA234 sample.(data are not shown).

When having and draw the curve of average serum concentration relative to the time without when the data of 504 hours to all DRA233PK time points.The serum-concentration of DRA234 is visible in Figure 18 C relative to the curve of time.Results expression is observed data collection, and passes through WinNonLin ^tMcomputed in software predicated value.Rsq value and Rsq adjustable value are the goodness of fit statistic regulating the final elimination stage estimating the front and back of counting that HL_Lambda z uses.Only use can not the time point of anti-drug antibodies of detection level time, the approximate transformation period of DRA233 is 95 hours, and DRA234's is 84 hours.See Figure 18.

Come from the PK estimated value (in tabulated form) of WinNonLin program: by using WinNonlin ^tMprofessional software (v5.3), applies non-segmentation analysis, uses the precompile model 201 (even weighting) used for IV bolus to carry out pharmacokinetic analysis.Use linear trapezoidal rule, utilize linear/logarithm interpolation computing method to carry out estimated time 0 can measure concentration (T is last) serum concentration-time area under curve (AUC) to last.Estimate following pharmacokinetics configuration parameter: maximum observation serum-concentration (Cmax), finally can measure the concentration under concentration (C is last), reach the time (Tmax) of maximum observation serum-concentration, the apparent final elimination transformation period (HL λ z), the AUC (AUC is total) of concentration can be measured from administration time to last, infinitely-great AUC (AUCINF_obs) is passed in time from administration time, serum clearance rate (Cl_obs), based on volume of distribution (Vz_obs) and the average retention time (MRT) of terminal stage.See Figure 19 and 20.The present embodiment shows, and test pI variant having serum half-life and slower clearance rate in longer body compared with parent molecule DRA161 in mouse.

embodiment 5: dual specific homodimer molecule-AntiCD3 McAb and anti-PSMA

Use standard technique structure in conjunction with the dual specific homodimer of CD3 and PSMA.These constructs, namely TSC249, TSC250, TSC251, TSC252, TSC295, TSC296, TSC301 and TSC302 are described in table 5.By realizing inserting N-terminal scFv binding domain by restriction enzyme HindIII and Xhol or Agel and Xhol digestion parent template and scFv inset, differentiating desired fragment, and being separated by agarose gel purification, and connecting.By digesting parent template with limiting enzyme EcoRI and Notl and scFv inset realizes inserting C-terminal scFv binding domain, differentiating desired fragment, and being separated by agarose gel purification, and connecting.

Table 5: Binding peptide sequence and component

embodiment 6: dual specific homodimer molecule-AntiCD3 McAb and anti-RON

Standard technique is used to build the bispecific molecule of the homodimer had in conjunction with CD3 and RON.These constructs, i.e. TSC275, TSC277, TSC278 and TSC279, be described in table 6.TSC275 contains VL and the VH chain of hu4C04, is respectively SEQ IDNO:187 and 188, and these chains are parts of RON binding domain.Except sudden change/replacement indicated in such as table 6, TSC277, TSC278 are identical with TSC279 and TSC275.

Table 6: Binding peptide sequence and complementary sequence

embodiment 7: dual specific homodimer molecule-AntiCD3 McAb and anti-CD19

Standard technique is used to build the bispecific molecule of the homodimer had in conjunction with CD3 and CD19, such as, see PCT publication No. WO2007/146968.These constructs, i.e. TSC233, TSC234, TSC235, TSC240, TSC241 and TSC242, be described in table 7.TSC233 contains VL (amino acid/11-111 of SEQ ID NO:196) and the VH (the amino acid/11 28-251 of SEQ ID NO:196) of HD37 chain, and these chains are parts of CD19 binding domain.

Table 7: Binding peptide sequence and component

embodiment 8: dual specific heterodimer molecule-AntiCD3 McAb and anti-CD19

To be similar to the mode described in PCT announcement WO2011/090762, by carrying out common transfection to build to the nucleic acid of coding as aminoacid sequence indicated in table 8 the different heterodimeric bispecific molecule comprising CD19 binding domain and CD3 binding domain.The polypeptide chain different by coexpression two manufactures heterodimer, and polypeptide chain comprises immunoglobulin (Ig) CH1 heterodimeric territory and another polypeptide chain comprises immunoglobulin (Ig) CL heterodimeric territory.

Table 8: the common transfection built for AntiCD3 McAb and anti-CD19 heterodimer

* the amino acid/11-244 of SEQ ID NO:10

The amino acid/11-244 of * SEQ ID NO:8

The amino acid/11-247 of * * SEQ ID NO:14

TSC049 comprises from its N-terminal to C-terminal: HD37 (anti-CD19) scFv, human IgG1 SCC-P hinge, human IgG1 CH2, human IgG1 CH3 and people CH1.TSC096 comprises from its N-terminal to C-terminal: HD37 (anti-CD19) scFv, human IgG1 SCC-P hinge, human IgG1 CH2, human IgG1 CH3 and people C κ (YAE).TSC125 comprises from its N-terminal to C-terminal: Cris7 (AntiCD3 McAb) scFv, human IgG1 SCC-P hinge, human IgG1 CH2, human IgG1 CH3 and people CH1.TSC218 comprises from its N-terminal to C-terminal: DRA221 (AntiCD3 McAb) scFv, human IgG1 SCC-P hinge, human IgG1 CH2, human IgG1 CH3 and people CH1.TSC219 comprises from its N-terminal to C-terminal: DRA222 (AntiCD3 McAb) scFv, human IgG1 SCC-P hinge, human IgG1 CH2, human IgG1 CH3 and people CH1.TSC220 comprises from its N-terminal to C-terminal: DRA224 (AntiCD3 McAb) scFv, human IgG1 SCC-P hinge, human IgG1 CH2, human IgG1 CH3 and people CH1.TSC221 comprises from its N-terminal to C-terminal: DRA221 (AntiCD3 McAb) scFv, human IgG1 SCC-P hinge, human IgG1 CH2, human IgG1 CH3 and people C κ (YAE).TSC222 comprises from its N-terminal to C-terminal: DRA222 (AntiCD3 McAb) scFv, human IgG1 SCC-P hinge, human IgG1 CH2, human IgG1 CH3 and people C κ (YAE).TSC223 comprises from its N-terminal to C-terminal: DRA224 (AntiCD3 McAb) scFv, human IgG1 SCC-P hinge, human IgG1 CH2, human IgG1 CH3 and people C κ (YAE).

embodiment 9: dual specific heterodimer molecule-AntiCD3 McAb and anti-PSMA

To be similar to the mode described in PCT announcement WO2011/090762, by carrying out common transfection to build to the nucleic acid of coding as aminoacid sequence indicated in table 9 the different heterodimeric bispecific molecule comprising PSMA binding domain and CD3 binding domain.The polypeptide chain different by coexpression two manufactures heterodimer, and polypeptide chain comprises immunoglobulin (Ig) CH1 heterodimeric territory and another polypeptide chain comprises immunoglobulin (Ig) CL heterodimeric territory.Some PSMA binding domain are described in PCT publication No. WO2011/090761.

Table 9: the cotransfection built for AntiCD3 McAb and anti-PSMA heterodimer

* the amino acid/11-244 of SEQ ID NO:8

The amino acid/11-243 of * SEQ ID NO:216

The amino acid/11-243 of * * SEQ ID NO:226

" humanization 107-1A4VL-VH#_2scFv " and " humanization 107-1A4VL-VH#_1scFv " is the humanization scFv based on anti-human PSMA 107-1A4 monoclonal antibody, and is also described in PCT publication No. WO2011/090761." humanization 107-1A4VL-VH#2scFv " and " humanization 107-1A4VL-VH#1scFv " corresponds respectively to the amino acid/11-243 of SEQ ID NO:216 and 226.

TSC192 comprises from its N-terminal to C-terminal: humanization 107-1A4 (anti-PSMA) VL-VH#2scFv, human IgG1 SCC-P hinge, human IgG1 CH2, human IgG1 CH3 and people C κ (YAE).TSC195 comprises from its N-terminal to C-terminal: humanization 107-1A4 (anti-PSMA) VL-VH#2scFv, human IgG1 SCC-P hinge, human IgG1 CH2, human IgG1 CH3 and people CH1.TSC193 comprises from its N-terminal to C-terminal: humanization 107-1A4 (anti-PSMA) VL-VH#1scFv, human IgG1 SCC-P hinge, human IgG1 CH2, human IgG1 CH3 and people C κ (YAE).

TSC219 comprises from its N-terminal to C-terminal: DRA222 (AntiCD3 McAb) scFv, human IgG1 SCC-P hinge, human IgG1 CH2, human IgG1 CH3 and people CH1.TSC222 comprises from its N-terminal to C-terminal: DRA222 (AntiCD3 McAb) scFv, human IgG1 SCC-P hinge, human IgG1 CH2, human IgG1 CH3 and people C κ (YAE).TSC254 comprises from its N-terminal to C-terminal: humanization 107-1A4 (anti-PSMA) VL-VH#1scFv, human IgG1 SCC-P hinge, human IgG1 CH2, human IgG1 CH3, connexon and people CH1.TSC258 comprises from its N-terminal to C-terminal: DRA222 (AntiCD3 McAb) scFv, human IgG1 SCC-P hinge, human IgG1 CH2, human IgG1 CH3, connexon and people C κ (YAE).

embodiment 10: the polypeptide heterodimer of target CD19 and the target caused by homodimer dependent T cell is bred

In order to the effect of more different dual specific polypeptide heterodimer molecules in the dependent T cell activation of induction target and propagation, compare three kinds of different homologous dimerization bispecific molecule (TSC129a, TSC233 and TSC234 (is SEQ ID NO:245 respectively, 196 and 198)), they have common anti-CD19 binding domain (HD37) the AntiCD3 McAb binding domain different with three, and (that TSC129a is Cris7, TSC233's is the scFv binding domain (amino acid/11-244 of SEQ ID NO:10 coming from DRA221, TSC234's is the scFv binding domain (amino acid/11-244 of SEQ ID NO:8) coming from DRA222).In addition, compare three kinds of different heterodimeric bispecific molecules (TSC127, TSC227, TSC228), they have three different AntiCD3 McAb binding domain, and (that TSC127 is Cris7, TSC227's is the scFv binding domain (amino acid/11-244 of SEQ ID NO:10) coming from DRA221, and TSC228's is the scFv binding domain (amino acid/11-244 of SEQ ID NO:8) coming from DRA222).

Obtain Daudi burkitt's lymphoma cell (CD19+) from ATCC (Manassas, VA) and cultivate according to provided scheme.Use standard ficoll gradient is separated peripheral blood mononuclear cell (PBMC) from human blood.The cell of separation is washed in normal saline buffer solution.Use the elutriation T cell separating kit II being obtained from Miltenyi Biotec (Bergisch Gladbach, Germany) in addition, use manufacturers's scheme to be separated T cell from these PBMC.

Propagation is assessed by the T cell colony be separated with Fluoresceincarboxylic acid oxalic acid succinimide ester (CFSE) mark.By the T cell that marks through CFSE with 100,000 T cell/hole and 33,000 tumour cell/hole are seeded in 96 orifice plates at the bottom of U-shaped, are about 3:1 to realize T cell to tumour cell ratio.The test molecule of concentration in 1nM to 5fM scope is joined in the cell mixture that 200uL/ hole is altogether supplemented with in RPMI 1640 substratum of 10% people or bovine serum, Sodium.alpha.-ketopropionate and non-essential amino acid.At 37 DEG C, 5%CO in moistening couveuse ₂under plate is hatched.After 3 days, with antibody labeled cells to carry out flow cytometry.Labeled cell and in its raw sheet washing in case by transfer during loss cell minimize, and containing 0.2% bovine serum albumin normal saline buffer solution in carry out all marks.First, at room temperature by cell preincubate 15 minutes together with 100ug/ml human IgG.Subsequently, cell is hatched together with the mixture (cumulative volume 50ul) of the antibody through following dye marker: CD5-PE, CD4-APC, CD8-Pacific Ocean is blue, CD25-PE-Cy7, and 7-aminoactinomycin D (being after this 7AAD), continue 40 minutes.Washed twice by plate, settling flux is in 80 to 120ul volumes and on BD LSRII flow cytometer, operate 80% inclusion obtaining each hole immediately.Use FlowJo software analysis sample file to calculate according to its CFSE curve negotiating CD4+ or CD8+T cell alive (being 7AAD-, CD5+CD25+CD4+ or 7AAD-CD5+CD25+CD8+ respectively) that lock choosing activates in order the per-cent and number that have carried out fissional cell at least one times.Use Microsoft Excel computed in software mean value and standard deviation.Use Microsoft Excel or Graphpad Prism curve plotting figure.

Show the analysis of CD4+ and the CD8+ colony alive of the Daudi cell come from through complete T-cell process, under showing that the Daudi cell of target CD19 antigen exists, total cellular score and proliferative cell per-cent all significantly increase (Figure 21 and 22).

For homologous dimerization dual specific polypeptide (TSC129a, TSC233, TSC234), the propagation between CD4+ and CD8+ cell quite (Figure 21).The propagation between CD4+ and CD8+ cell suitable (Figure 21) of TSC129a and TSC234 induction, the pI variant AntiCD3 McAb scFv binding domain and the parent Cris7 binding domain that show to come from DRA222 have activity equivalent in fact.T cell growth curve and TSC129a and TSC234 of TSC233 induction are slightly different, and the CD8+ propagation that the CD4+ propagation of wherein inducing reduces (Figure 21 A) and induces increases (Figure 21 B) a little.This pI variant AntiCD3 McAb scFv binding domain showing to come from DRA221 in active with parent Cris7 binding domain imcomplete equivalent.

For heterodimeric dual specific polypeptide (TSC127, TSC227, TSC228), under Daudi cell exists, the propagation of CD8+T cell is higher than CD4+T cell (Figure 22).The propagation between CD4+ and CD8+ cell suitable (Figure 22) of TSC127 and TSC228 induction, the pI variant AntiCD3 McAb scFv binding domain and the parent Cris7 binding domain that show to come from DRA222 have activity equivalent in fact.T cell growth curve and TSC127 and TSC228 of TSC227 induction are slightly different, and the CD4+ propagation of wherein inducing reduces and the CD8+ of induction breeds quite.Again, this pI variant AntiCD3 McAb scFv binding domain showing to come from DRA221 in active with parent Cris7 binding domain imcomplete equivalent.

embodiment 11: the polypeptide heterodimer of target CD19 and the weight caused by homodimer directed T cell cytotoxicity

In order to the effect of more different dual specific polypeptide heterodimer molecules in induction target dependent T cell cytotoxicity, compare three kinds of different homologous dimerization bispecific molecule (TSC129a, TSC233 and TSC234 (is SEQ ID NO:245 respectively, 196 and 198)), they have common anti-CD19 binding domain (HD37) the AntiCD3 McAb binding domain different with three, and (that TSC129a is Cris7, TSC233's is the scFv binding domain (amino acid/11-244 of SEQ ID NO:10 coming from DRA221, TSC234's is the scFv binding domain (amino acid/11-244 of SEQ ID NO:8) coming from DRA222).In addition, compare three kinds of different heterodimeric bispecific molecules (TSC127, TSC227, TSC228), they have three different AntiCD3 McAb binding domain, and (that TSC127 is Cris7, TSC227's is the scFv binding domain (amino acid/11-244 of SEQ ID NO:10) coming from DRA221, and TSC228's is the scFv binding domain (amino acid/11-244 of SEQ ID NO:8) coming from DRA222).

Obtain Daudi burkitt's lymphoma cell (CD19+) from ATCC (Manassas, VA) and cultivate according to provided scheme.Use standard ficoll gradient is separated peripheral blood mononuclear cell (PBMC) and washs in normal saline buffer solution from human blood.Use the elutriation T cell separating kit II being obtained from Miltenyi Biotec (Bergisch Gladbach, Germany) in addition, use manufacturers's scheme to be separated T cell from PBMC.

Pass through ⁵¹cr release measures assessment cytotoxicity.Use 0.3mCi ⁵¹cr process about 5 × 10 ⁶individual Daudi cell, and 75 minutes are hatched at 37 DEG C.After 75 min, with substratum (RPMI+10%FBS) by cell washing 3 times, and settling flux is in 11.5mL substratum.(about 20,000 cells/well) in 96 hole U-shaped base plates are assigned to every hole 50 μ L by this suspension.The bispecific molecule of concentration in 500pM to 0.1pM scope is added Daudi cell, makes cumulative volume reach 100 μ L/ holes.At room temperature target cell is hatched 15 minutes.Then the T cell (about 200,000) of 100 μ L separation is added to make T cell reach 10:1 to target cell ratio.50 μ L 0.8%NP-40 are added the control wells containing target cell, leaves standstill 15 minutes, then add 100 μ L substratum and dissolve contrast completely to provide.

Plate is hatched 4 hours, then rotate 3 minutes with 1500rpm, and from each hole transferase 12 5 μ L supernatant liquor to the corresponding aperture of 96 hole Luma sample panel.Allow sample panel in chemical safety cover air-dry 18 hours, on Topcount scintillometer, then use standard scheme to read radioactivity.

The analysis of cell toxicity data is shown to the T cell targeted cytotoxic (Figure 23) obtained with Daudi cell under T cell and the directed bispecific molecule of anti-CD19 exist.

For homologous dimerization dual specific polypeptide (TSC129a, TSC233, TSC234), observation cytotoxicity between TSC129a and TSC234 is suitable (Figure 23 A), shows that this DRA222pI variant and parent Cris7 binding domain have equivalence active.Under TSC233 exists, observe that cytotoxicity declines a little, show that the activity of this DRA221pI variant is lower than parent Cris7 binding domain.

For heterodimeric dual specific polypeptide (TSC127, TSC227, TSC228), observation cytotoxicity between TSC127 and TSC228 is suitable (Figure 23 B), shows that this DRA222scFv binding domain pI variant and parent Cris7 binding domain have equivalence active.Under TSC227 exists, observe that cytotoxicity declines a little, show that the activity of this DRA221scFv binding domain pI variant is lower than parent Cris7 binding domain.

embodiment 12: the redirected T cell caused by the homologous peptide dimer of target RON is thin cellular toxicity

In order to the effect of more different dual specific polypeptide heterodimer molecules in induction target dependent T cell cytotoxicity, compare four kinds of different homologous dimerization bispecific molecule (TSC275, TSC277, TSC278 and TSC279), they have common AntiCD3 McAb binding domain (coming from the scFv binding domain (amino acid/11-244 of SEQ ID NO:8) of DRA222) and (that TSC275 is hu4C04scFv with four variants of primary antibodie RON binding domain, that TSC277 is hu4C04scFv (A43K/Q240E), that TSC278 is hu4C04scFv (A43T), and that TSC279 is hu4C04scFv (Q165R)).

Obtain MDA-MB-453 breast cancer cell (RON+) from ATCC (Manassas, VA) and cultivate according to the scheme that ATCC provides.Use standard ficoll gradient is separated peripheral blood mononuclear cell (PBMC) and washs in normal saline buffer solution from human blood.Use the elutriation T cell separating kit II being obtained from Miltenyi Biotec (Bergisch Gladbach, Germany) in addition, use manufacturers's scheme to be separated T cell from PBMC.

Pass through ⁵¹cr release measures assessment cytotoxicity.Use 0.3mCi ⁵¹cr process about 5 × 10 ⁶individual Daudi cell, and 75 minutes are hatched at 37 DEG C.After 75 min, with substratum (RPMI+10%FBS) by cell washing 3 times, and settling flux is in 11.5mL substratum.(about 20,000 cells/well) in 96 hole U-shaped base plates are assigned to every hole 50 μ L by this suspension.The bispecific molecule of concentration in 10nM to 0.005fM scope is added Daudi cell, makes cumulative volume reach 100 μ L/ holes.At room temperature target cell is hatched 15 minutes.Then the T cell (about 100,000) of 100 μ L separation is added to make T cell reach 5:1 to target cell ratio.50 μ L 0.8%NP-40 are added the control wells containing target cell, leaves standstill 15 minutes, then add 100 μ L substratum and dissolve contrast completely to provide.

Plate is hatched 24 hours, then rotate 3 minutes with 1500rpm, and from each hole transferase 12 5 μ L supernatant liquor to the corresponding aperture of 96 hole Luma sample panel.Allow sample panel in chemical safety cover air-dry 18 hours, on Topcount scintillometer, then use standard scheme to read radioactivity.

The analysis of cell toxicity data is shown to the T cell targeted cytotoxic (Figure 24) obtained with MDA-MB-453 cell under T cell and the directed bispecific molecule of anti-RON exist.

For homologous dimerization dual specific polypeptide (TSC275, TSC277, TSC278 and TSC279), observation cytotoxicity generally suitable (Figure 24), shows the sudden change not remarkably influenced general activity in ORN196 binding domain.But the maximum dissolving observed for TSC278 is slightly lower than other three kinds of variants (Figure 24).Maximum dissolving has been observed between the concentration of 10pM and 10nM.

embodiment 13: the T cell knot of dual specific polypeptide heterodimer and homodimer close

In order to compare with pI variant CD3 binding domain (scFv (amino acid/11-244 of SEQ IDNO:8) found in DRA222) the dual specific peptide molecule that is feature in conjunction with the effect in T cell, anti-CD19 × AntiCD3 McAb dual specific polypeptide heterodimer TSC228 is compared (as embodiment 8 in two independently experiment, described in table 8) cell in conjunction with on the cell of feature and anti-PSMA × AntiCD3 McAb dual specific homologous peptide dimer TSC249 (as embodiment 5, described in table 5) in conjunction with feature.

Jurkat (CD3 is obtained from ATCC (Manassas, VA) ⁺) T cell leukemia cell and cultivating according to the scheme that ATCC provides.By at 4 DEG C by 5 × 10 ⁵individual Jurkat cell hatches 30 minutes to assess combination with the concentration of 250nM to 0.125nM (TSC249) or 125nM to 1pM (TSC228) together with bispecific molecule TSC228 or TSC249 of serial dilution.By cell washing three times, then at 4 DEG C, hatch 30 minutes again together with Goat anti human IgG-FITC (1:200 extent of dilution).Then cell is washed three times again, in 1% polyoxymethylene fixing and on FACS-Calibur instrument reading.

At FlowJo v7.5 (Tree Star, Inc., Ashland, OR) in analysis that high FSC, high SSC subset are carried out show bispecific molecule TSC228 and TSC249 and be combined with the dose-dependently of Jurkat cell (being Figure 25 A and B respectively).The TSC228 dual specific polypeptide heterodimer only having an AntiCD3 McAb binding domain in conjunction with time apparent avidity (EC ₅₀=24.5nM) lower than the TSC249 dual specific homologous peptide dimer (EC having two AntiCD3 McAb binding domain ₅₀=1.9nM).This shows that TSC249 dual specific polypeptide heterodimer has certain binding affinity.

embodiment 14: the target caused by dual specific homologous peptide dimer of target PSMA is complied with rely property T cell propagation

In order to measure the effect of different dual specific polypeptide heterodimer molecules in the dependent T cell activation of induction target and propagation, when there is not target cell, expressing the homologous dimerization bispecific molecule (TSC249 prostate cancer cell line of PSMA, the prostate cancer cell line of not expressing PSMA and T cell being tested there is anti-PSMA binding domain (hu107-1A4) and AntiCD3 McAb binding domain (coming from the scFv (amino acid/11-244 of SEQ ID NO:8) of DRA222), see embodiment 5, table 5).

Obtain C4-2B prostate cancer cell (PSMA+) from Maryland Anderson Cancer center (Houston, TX) and cultivate according to provided scheme.DU-145 prostate cancer cell (PSMA is obtained from ATCC (Manassas, VA) ^-) and cultivate according to provided scheme.Use standard ficoll gradient is separated peripheral blood mononuclear cell (PBMC) and washs in normal saline buffer solution further from human blood.Then use the elutriation T cell separating kit II being obtained from Miltenyi Biotec (Bergisch Gladbach, Germany), use manufacturers's scheme to be separated T cell from these PBMC.

Propagation is assessed by the T cell colony be separated with Fluoresceincarboxylic acid oxalic acid succinimide ester (CFSE) mark.Set up three independent experiments simultaneously.By the T cell that marks through CFSE only with TSC249 molecular mixing; By the T cell that marks through CFSE and DU-145 cell and TSC249 molecular mixing; With the T cell will marked through CFSE and C4-2B cell and TSC249 molecular mixing.By the T cell that marks through CFSE with 100,000 cells/well and 33,000 tumour cell/hole are seeded in 96 orifice plates at the bottom of U-shaped, are approximately 3:1 to realize T cell to tumour cell ratio.The test molecule of concentration in 20nM to 5fM scope is joined in the cell mixture in RPMI 1640 substratum being supplemented with 10% people or bovine serum, Sodium.alpha.-ketopropionate and non-essential amino acid in 200uL/ hole altogether.At 37 DEG C, 5%CO in moistening couveuse ₂under plate is hatched.After 3 days, with antibody labeled cells to carry out flow cytometry.Labeled cell and in its raw sheet washing in case by transfer during loss cell minimize, and containing 0.2% bovine serum albumin normal saline buffer solution in carry out all marks.First, at room temperature by cell preincubate 15 minutes together with 100ug/ml human IgG.Subsequently, cell is hatched together with the mixture (cumulative volume 50ul) of the antibody through following dye marker: CD5-PE, CD4-APC, CD8-Pacific Ocean is blue, CD25-PE-Cy7, and 7-aminoactinomycin D (being after this 7AAD), continue 40 minutes.Washed twice by plate, settling flux is in 80 to 120ul volumes and on BD LSRII flow cytometer, operate 80% inclusion obtaining each hole immediately.Use FlowJo software analysis sample file to calculate according to its CFSE curve negotiating CD4+ or CD8+T cell alive (being 7AAD-, CD5+CD25+CD4+ or 7AAD-CD5+CD25+CD8+ respectively) that lock choosing activates in order the per-cent and number that have carried out fissional cell at least one times.Use Microsoft Excel computed in software mean value and standard deviation.Use Microsoft Excel or Graphpad Prism curve plotting figure.

Show the analysis of CD4+ and the CD8+ colony alive of the C4-2B cell come from through complete T-cell process, under existing with the C4-2B cell of expressing target PSMA antigen after TSC249 process, total cellular score significantly increases (Figure 26 A).This increase (Figure 26 B) is not seen under the DU-145 cell of not expressing target PSMA antigen after with TSC249 process exists.Finally, this increase (Figure 26 C) is not seen yet when the T cell be separated with TSC249 process.Which demonstrate the target dependency of TSC249 T cell propagation of induction under the tumour cell of expressing target antigen exists.

embodiment 15: the dual specific target of body build-in test CD3 and PSMA

In order to confirm the effect of AntiCD3 McAb and anti-PSMA bispecific molecule Tumor suppression growth aspect in vivo, following any one or many persons can be used to assess PSMA oriented molecule.

Preventative process or prevention to the tumour transplatation of Subcutaneous tumor: the tumor cell line (such as LNCaP, LNCaP C4-2, LNCaP C4-2B, VCaP, CWR22Rv1, LAPC4, MDA-PCa-2b, LuCaP 23.1, LuCaP 58, LuCaP 70, LuCaP 77) of cultivated expression PSMA is mixed with human lymphocyte (T cell of people's peripheral blood mononuclear cell or purifying), and subcutaneous injection is in immunodeficient mouse (such as SCID, NOD/SCID etc.).Injection the same day and some skies subsequently through the anti-PSMA bispecific molecule of intravenous injection.As by gross tumor volume assessed, the tumour of molecule to expression in vivo PSMA corresponding to the dose-dependent inhibition instruction grown tumour has effect.

To the therapeutic treatment of the Subcutaneous tumor previously determined or disappear: by tumor cell line (such as LNCaP, LNCaP C4-2, LNCaP C4-2B, VCaP, CWR22Rv1, LAPC4, MDA-PCa-2b, LuCaP 23.1AI, LuCaP 58, LuCaP 70, the LuCaP 77) subcutaneous injection of cultivated expression PSMA in immunodeficient mouse (such as SCID, NOD/SCID etc.).Monitoring tumor growth, and begin one's study when tumour display clearly grows the sign of (typically, volume is about 200mm3).Injecting the same day, through intravenous injection human lymphocyte (T cell of people's peripheral blood mononuclear cell or purifying) and anti-PSMA bispecific molecule.Within subsequently some days, inject anti-PSMA bispecific molecule.As by gross tumor volume assessed, the molecule tumour to expression in vivo PSMA corresponding to the instruction of the dose-dependent inhibition of tumor growth has effect.

Prophylactic treatment or prevention to the tumour transplatation of tumour in shin bone: the tumor cell line (such as LNCaP C4-2, LNCaP C4-2B, VCaP, CWR22Rv1, LAPC4, MDA-PCa-2b, LuCaP 23.1, LuCaP 58, LuCaP70, LuCaP 77) of cultivated expression PSMA is mixed with human lymphocyte (T cell of people's peripheral blood mononuclear cell or purifying), and in shin bone, injects immunodeficient mouse (such as SCID, NOD/SCID etc.).Injection the same day and some skies subsequently through the anti-PSMA bispecific molecule of intravenous injection.As entrusted observed value institute to be assessed by other of serum biomarker thing, roentgenogpaphy, fluorescence imaging, body weight loss and gross tumor volume, to the tumour of molecule to expression in vivo PSMA of the dose-dependent inhibition of tumor growth instruction correspondence, there is effect.

To the therapeutic treatment of tumour in the shin bone previously determined or disappear: by the tumor cell line (such as LNCaP C4-2, LNCaP C4-2B, VCaP, CWR22Rv1, LAPC4, MDA-PCa-2b, LuCaP 23.1AI, LuCaP 58, LuCaP70 and LuCaP 77) of cultivated expression PSMA through intratibia injection in immunodeficient mouse (such as SCID, NOD/SCID etc.).Monitoring tumor growth, and begin one's study when tumour display clearly grows the sign of (typically, volume is about 200mm3).Injecting the same day, through intravenous injection human lymphocyte (T cell of people's peripheral blood mononuclear cell or purifying) and anti-PSMA bispecific molecule.Within subsequently some days, inject anti-PSMA bispecific molecule.As entrusted observed value institute to be assessed by other of serum biomarker thing, roentgenogpaphy, fluorescence imaging, body weight loss and gross tumor volume, to the tumour of molecule to expression in vivo PSMA of the dose-dependent inhibition of tumor growth instruction correspondence, there is effect.

embodiment 16: caused by the polypeptide heterodimer of target PSMA and homodimer be redirected T cell cytotoxicity

In order to the effect of more different dual specific polypeptide heterodimer molecules in induction target dependent T cell cytotoxicity, compare two kinds of different homologous dimerization bispecific molecules (TSC194, TSC249), they have common anti-PSMA binding domain (hu107-1A4) the AntiCD3 McAb binding domain different with two (TSC194 be Cris7scFV, TSC249 be the scFv coming from DRA222).TSC194 is homologous dimerization dual specific (huVL-VH#2107-1A4scFv-Fc-Cris7scFv) (SEQ ID NO:250 and 251).

Obtain C4-2B cell (PSMA+) from Maryland Anderson and add in 10%FBS according to disclosed culture condition cultivate at RPMI-1640 substratum (Life Technologies, Carlsbad, CA).Use standard ficoll gradient is separated peripheral blood mononuclear cell (PBMC) and washs in normal saline buffer solution from human blood.Use the elutriation T cell separating kit II being obtained from Miltenyi Biotec (Bergisch Gladbach, Germany) in addition, use manufacturers's scheme to be separated T cell from PBMC.

Pass through ⁵¹cr release measures assessment cytotoxicity.Use 0.3mCi ⁵¹cr process about 5 × 10 ⁶individual C4-2 cell, and 75 minutes are hatched at 37 DEG C.After 75 min, with substratum (RPMI+10%FBS) by cell washing 3 times, and settling flux is in 11.5mL substratum.(about 20,000 cells/well) in 96 hole U-shaped base plates are assigned to every hole 50 μ L by this suspension.The bispecific molecule of concentration in 500pM to 0.2pM scope is added C4-2 cell, makes cumulative volume reach 100 μ L/ holes.At room temperature target cell is hatched 15 minutes.Then the T cell (about 200,000) of 100 μ L separation is added to make T cell reach 10:1 to target cell ratio.50 μ L 0.8%NP-40 are added the control wells containing target cell, leaves standstill 15 minutes, then add 100 μ L substratum and dissolve contrast completely to provide.

Plate is hatched 4 hours, then rotate 3 minutes with 1500rpm, and from each hole transferase 12 5 μ L supernatant liquor to the corresponding aperture of 96 hole Luma sample panel.Allow sample panel in chemical safety cover air-dry 18 hours, and then Topcount scintillometer uses standard scheme read radioactivity.

The analysis of cell toxicity data is shown to the T cell targeted cytotoxic (Figure 27) obtained with C4-2 cell under T cell and the directed bispecific molecule of anti-PSMA exist.For homologous dimerization dual specific polypeptide (TSC194, TSC249), observation cytotoxicity between TSC194 and TSC249 is suitable (table 10), shows that this DRA222 AntiCD3 McAb scFv binding domain pI variant and parent Cris7 binding domain have equivalence active.

Table 10: the cytotoxicity observed

	The EC of RTCC ₅₀
		TSC194	2.8±0.6pM
TSC249	2.9±1.0pM

Claims

1. a CD3 Binding peptide, it comprises the CD3 binding domain compared with having the binding domain of aminoacid sequence SEQ ID NO:41 with the iso-electric point of reduction.

2. CD3 Binding peptide as claimed in claim 1, wherein said binding domain comprises VH district and VL district, and wherein said VH district and described VL district comprise framework region.

3. CD3 Binding peptide as claimed in claim 2, wherein can by replacing the amino acid of positively charged with neutral amino acids and/or modifying two or more amino acid in the framework region of VH and/or VL with electronegative aminoacid replacement neutral amino acids.

4. CD3 Binding peptide as claimed in claim 3, the modified amino acid of wherein said framework region is by aminoacid replacement contained in the human germ-line system IgG sequence of correspondence.

5. CD3 Binding peptide as claimed in claim 4, wherein said human germ-line system IgG sequence comprises SEQ ID NO:43

6. CD3 Binding peptide as claimed in claim 4, wherein said human germ-line system IgG sequence comprises SEQ ID NO:44

7. the CD3 Binding peptide as described in Claim 1-3, also comprises front hinge area.

8. CD3 Binding peptide as claimed in claim 7, wherein said front hinge area has the iso-electric point of reduction compared with having the CD3 Binding peptide of the front hinge area of RRT.

9. a CD3 Binding peptide, it comprises the CD3 binding domain with framework region, front hinge area and hinge area,

Wherein said CD3 Binding peptide has three or more modified amino acid compared with SEQ ID NO:42, described modification be in described framework region and/or front hinge area with neutral amino acids replace positively charged amino acid and/or with electronegative aminoacid replacement neutral amino acids.

10. CD3 Binding peptide as claimed in claim 9, wherein can modify at least two or more amino acid in the framework region of VH and/or VL by the amino acid with neutral amino acids replacement positively charged and/or with electronegative aminoacid replacement neutral amino acids.

11. CD3 Binding peptides as described in claim 3 and 9, wherein can by replacing the amino acid of positively charged and/or modifying an at least four or more amino acid in the framework region of VH and/or VL with electronegative aminoacid replacement neutral amino acids with neutral amino acids.

12. CD3 Binding peptides as described in claim 3 and 9, wherein can by replacing the amino acid of positively charged with neutral amino acids and/or modifying to five amino acid three in the framework region of VH and/or VL with electronegative aminoacid replacement neutral amino acids.

13. CD3 Binding peptides as described in claim 3 and 9, wherein can by replacing the amino acid of positively charged with neutral amino acids and/or modifying three to ten amino acid in the framework region of VH and/or VL with electronegative aminoacid replacement neutral amino acids.

14. CD3 Binding peptides as described in claim 8 to 9, the wherein said front hinge area of CD3 Binding peptide comprises aminoacid sequence SSS or SST.

15. CD3 Binding peptides as described in claim 1 to 8, wherein said CD3 Binding peptide also comprises hinge area and constant region.

16. CD3 Binding peptides as described in claim 1 to 15, wherein said CD3 Binding peptide has the experience iso-electric point of at least 1 pI unit less of polypeptide SEQ ID NO:4.

17. CD3 Binding peptides as claimed in claim 1, wherein said CD3 Binding peptide is humanized antibody, scFv (scFv) or SMIP.

18. CD3 Binding peptides as claimed in claim 15, wherein said CD3 Binding peptide comprises CD3 binding domain, hinge area and constant region from N-terminal to C-terminal.

19. CD3 Binding peptides as claimed in claim 15, wherein said Binding peptide comprises constant region, hinge area and CD3 binding domain from N-terminal to C-terminal.

20. CD3 Binding peptides as claimed in claim 15, wherein said Binding peptide also comprises the second binding domain.

21. CD3 Binding peptides as claimed in claim 20, wherein said Binding peptide comprises CD3 binding domain, hinge area, constant region and the second binding domain from N-terminal to C-terminal.

22. CD3 Binding peptides as claimed in claim 20, wherein said CD3 Binding peptide comprises the second binding domain, hinge area, constant region and CD3 binding domain from N-terminal to C-terminal.

23. CD3 Binding peptides as claimed in claim 15, wherein said CD3 Binding peptide also comprises heterodimeric territory.

24. CD3 Binding peptides as claimed in claim 23, wherein said CD3 Binding peptide comprises CD3 binding domain, hinge area, constant region and heterodimeric territory from N-terminal to C-terminal.

25. CD3 Binding peptides as described in claim 1 to 24, wherein said CD3 Binding peptide also comprises the second binding domain.

26. CD3 Binding peptides as claimed in claim 25, wherein said second binding domain binding target molecule or with its interaction, and described CD3 Binding peptide inducing T cell cytotoxicity.

27. CD3 Binding peptides as claimed in claim 26, wherein said second binding domain in conjunction with tumor associated antigen or with its interaction.

28. CD3 Binding peptides as claimed in claim 27, wherein said CD3 Binding peptide inducing T cell dissolves tumour cell.

29. CD3 Binding peptides as claimed in claim 27, wherein said CD3 Binding peptide is in tumor vicinity induction polyclone T cell activation and amplification.

30. CD3 Binding peptides as described in claim 27 to 29, wherein said tumor associated antigen is selected from RON, c-Met, CEACAM-6, PSMA, EpCAM, CEA, PCTA-1, STEAP-1, STEAP-2, PSCA, PSA, PAP, ALCAM (CD166), PECAM-1, EphA2, CD151, CA-125/MUC16, MUC-1, MAGE-1, TROP2, IGF1R, TGFBR2, GHRHR, GHR, IL-6R, gp130, TNFR2, OSMR β, Patched-1, Frizzled, Robo1, LT β R, CD25, CD26, CD27, CD30, CD33, CD44, CD44v6, CD63, CD80, CD81, CD86, CD100, CD151, CXCR4, CCR5, HER-2/ErbB1, HER-3/ErbB3, HER-4/ErbB4, EGFR/ErbB1, EGFRvIII isotype, MUC2, MUC3, MUC4, MUC5 _aC, MUC5 _b, MUC7, β hCG, Lewis-Y, Ganglioside, GD3,9-O-ethanoyl-GD3, GM2, Globo H, Fucose acyl GM1, Poly SA, GD2, carbonic anhydrase IX (MN/CA IX), Sonic hedgehog (Shh), Wue-1, plasma cell antigen, (film combination) IgE, melanoma chondroitin sulfate protein-polysaccharide (MCSP), CCR8, TNF-α precursor, mesothelin, A33 antigen, Ly-6, desmoglein 4, the new epi-position of E-Calcium ionorphore, fetus acetylcholine receptor, CA19-9 marker, seedling Le Shi inhibitory substance (MIS) receptor II type, sTn (sialylated Tn antigen, TAG-72), FAP (inoblast active antigen), endosialin, LG, SAS, BCMA, TWEAKR/Fn14, FGFR4, VEGFR1, VEGFR2, SSX1 and SSX2.

31. CD3 Binding peptides as described in Claim 1-3 0, wherein said CD3 binding domain in conjunction with the tcr complex in T cell CD3 ε subunit or with its interaction.

32. CD3 Binding peptides as described in Claim 1-3 1, the internalization of wherein said CD3 Binding peptide inducing T cell receptor complex.

33. CD3 Binding peptides as described in Claim 1-3 2, wherein said CD3 binding domain is the humanization CD3 binding domain deriving from muroid Cris-7 monoclonal antibody or HuM291.

34. CD3 Binding peptides as claimed in claim 33, wherein said CD3 binding domain comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 of muroid Cris-7 antibody or HuM291.

35. CD3 Binding peptides as claimed in claim 1, wherein said CD3 binding domain comprises and is selected from SEQ ID NO:28, SEQ ID NO:30, the VH region sequence of SEQ ID NO:32, SEQ IDNO:38 and SEQ ID NO:40 or VL region sequence.

36. CD3 Binding peptides as claimed in claim 1, wherein said CD3 binding domain comprise be selected from SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30 and SEQ ID NO32 VH district and be selected from the VL district of SEQ ID NO:38 and SEQ ID NO:40.

37. CD3 Binding peptides as claimed in claim 1, wherein said CD3 binding domain comprises the VH district being selected from SEQ ID NO:28, SEQ ID NO:30 and SEQ ID NO:32 and the VL district being selected from SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38 and SEQ IDNO:40.

38. CD3 Binding peptides as claimed in claim 2, wherein said VH district comprises SEQ ID NO:28 and described VL district comprises SEQ ID NO:34.

39. CD3 Binding peptides as claimed in claim 2, wherein said VH district comprises SEQ ID NO:28 and described VL district comprises SEQ ID NO:38.

40. CD3 Binding peptides as claimed in claim 2, wherein said VH district comprises SEQ ID NO:26 and described VL district comprises SEQ ID NO:38.

41. CD3 Binding peptides as described in claim 9 and 14, wherein said VH district comprises SEQ ID NO:26 and described VL district comprises SEQ ID NO:34.

42. CD3 Binding peptides as claimed in claim 1, wherein said CD3 Binding peptide comprises the aminoacid sequence being selected from SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18 and SEQ IDNO20.

43. CD3 Binding peptides as claimed in claim 1, wherein said CD3 binding domain comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3, and wherein said heavy chain CDR3 comprises SEQ ID NO:51.

44. CD3 Binding peptide as claimed in claim 43, wherein heavy chain CDR1 comprises SEQ ID NO:49, and CDR2 comprises SEQ ID NO:50 and CDR3 comprises SEQ IDNO:51, and light chain CDR1 comprises SEQ ID NO:52, CDR2 comprises SEQ IDNO:53, and CDR3 comprises SEQ ID NO:54.

45. CD3 Binding peptides as claimed in claim 15, wherein constant region comprises CH2 and the CH3 territory of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD or its any combination; The immunoglobulin (Ig) CH3 territory of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, IgM or its any combination; IgE, IgM or its combination immunoglobulin (Ig) CH3 and CH4 territory.

46. CD3 Binding peptides as claimed in claim 15, wherein said constant region comprises CH2 and CH3.

47. CD3 Binding peptides as claimed in claim 15, wherein said constant region is made up of CH2 territory and CH3 territory in fact.

48. CD3 Binding peptides as described in claim 45 to 47, wherein said constant region is modified to reduce or eliminate effector function.

49. CD3 Binding peptides as described in claim 45 to 47, wherein said constant region is modified so that not complement-fixing.

50. CD3 Binding peptides as described in claim 45 to 47, wherein said constant region is modified so that not in conjunction with Fc γ acceptor.

51. CD3 Binding peptides as claimed in claim 50, wherein said Fc γ acceptor is selected from CD16, CD32 and CD64.

52. CD3 Binding peptides as claimed in claim 15, wherein said constant region comprises aminoacid sequence SEQ ID NO:55.

53. 1 kinds of nucleic acid, its any one polypeptide as described in claim 1 to 52 of encoding.

54. 1 kinds of nucleic acid be separated, comprise the nucleic acid being selected from SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQID NO:17, SEQ ID NO:19, SEQ ID NO:27 and SEQ ID NO:37.

55. 1 kinds of expression vectors, comprise the nucleic acid as described in claim 53 or 54.

56. 1 kinds of recombinant host cells, comprise expression vector as claimed in claim 32.

57. 1 kinds of compositions, comprise the CD3 Binding peptide according to any one of claim 1 to 52 and pharmaceutically acceptable supporting agent, thinner or vehicle.

58. 1 kinds of methods being used for the treatment of cancer or autoimmune disorder, comprise to the composition as claimed in claim 57 of patient therapeuticallv's significant quantity in need or the CD3 Binding peptide as described in claim 1 to 52.

59. CD3 Binding peptides as described in claim 1 to 52, wherein said CD3 Binding peptide forms dimer.

60. CD3 Binding peptides as claimed in claim 59, wherein said dimer is homodimer or heterodimer.

61. CD3 Binding peptides as claimed in claim 3, wherein compared with Cris-7VL J κ (Jk) district mouse sequence LQIT (SEQ ID NO:252), in the Jk district at least one amino acid modified Shi VL district.

62. CD3 Binding peptides as claimed in claim 61, wherein said Jk district comprises aminoacid sequence VEIK (SEQ ID NO:253).

63. CD3 Binding peptides as claimed in claim 25, wherein said second binding domain comprises and the aminoacid sequence being selected from following aminoacid sequence and having at least 90%, at least 95% or 100% identity: the amino acid/11-107 of SEQ ID NO:212 and 124-243; The amino acid/11-107 of SEQ IDNO:226 and 124-243; The amino acid/11-107 of SEQ ID NO:216 and 124-243; With 1-111 and 128-251 of SEQ ID NO:196.

64. CD3 Binding peptides as claimed in claim 1, wherein said Binding peptide comprises the aminoacid sequence with SEQ ID NO:57,59,61,63,65,67,69,71,186,190,192,194,196,198,200,202,204 or 206 with at least 90%, at least 95% or 100% identity.

65. CD3 Binding peptides as claimed in claim 1, wherein said polypeptide is a part for heterodimer.

66. CD3 Binding peptides as described in claim 65, wherein said heterodimer comprises a pair single chain polypeptide, wherein said strand is to comprising and being selected from following pairing and having at least 90%, the aminoacid sequence of at least 95% or 100% identity: SEQ ID NO:210 and 247, SEQ ID NO:210 and 218, SEQ ID NO:210 and 220, SEQ ID NO:208 and 249, SEQ ID NO:208 and 222, or SEQ ID NO:208 and 224, SEQ ID NO:212 and 218, SEQ ID NO:216 and 222, SEQ ID NO:228 and 226, or SEQ IDNO:214 and 218.

67. CD3 Binding peptides as claimed in claim 1, wherein said CD3 binding domain and SEQ ID NO:41 have about 90% to about 99% identity.